
T 



QassJLl 
Book ^ 



Copjyiigtit]^^ '-l^f 



COPYRIGHT DEPOSrr. 







i 



ESSENTIALS 



OP 



LABORATORY DIAGNOSIS 



DESIGNED FOR 
STUDENTS AND PRACTITIONERS ^ 



BY 

FRANCIS ASHLEY FAUGHT, M.D. 

FORMERLY DIRECTOR OF THE LABORATORY OF THE DEPARTMENT OF CLINICAL 

MEDICINE AND ASSISTANT TO THE PROFESSOR OF CLINICAL MEDICINE, 

MEDICO-CHIRURGICAL COLLEGE, ETC., PHILADELPHIA, PA. 



CONTAINING ELEVEN FULL-PAGE PLATES (FOUR IN COLORS) AND 
SElVENTY-EIGHT TEXT ENGRAVINGS 



SEVENTH REVISED AND ENLARGED EDITION 




PHILADELPHIA 

F. A. DAVIS COMPANY, Publishers 

1921 






IV''' 



COPYRIGHT, 1909 
COPYRIGHT, 1910 
COPYRIGHT, 1911 
COPYRIGHT, 1912 
COPYRIGHT, 1915 
COPYRIGHT, 1916 
COPYRIGHT, 1921 

BY 

F. A. DAVIS COMPANY 



Ccpyright, Great Britain. All Rights Reserved 



DEe-7'21 



PRESS OF 

F. A. DAVIS COMPANY 

PHILADELPHIA. U.S.A. 



©GI.A630679 



va J 



cr 



PREFACE TO SEVENTH EDITION. 



The clinical laboratory is no longer the simple collection of 
reagent bottles, test-tubes and alcohol-lamp of ten years ago. 
The development of many new methods in response to the de- 
mand for additional accurate information in the clinical study 
of cases has widened the scope of the clinical laboratory until 
it has now reached the dignity of a specialty of medicine. 

An effort to embody many of these new and valuable 
methods, found practical since the preparation of the former 
edition of this work, has forced the author to completely revise 
and to practically rewrite this book, and in so doing to depart 
in^ some measure from the original , restricted plan of the earlier 
editions. It is hope'd that the new methods introduced and the 
technic advised, while requiring considerable technical skill con- 
suming much time, will still remain within the chemical dex- 
terity of the average physician. 

In preparing this seventh edition, current literature has 
beetti carefully reviewed and the larger works on physiologic 
chemistry, medical chemistry and clinical diagnosis freely drawn 
upon, references to these have, whenever possible^ been inserted 
in the text. The author has depended much in the work of com- 
pilation upon Hawk's Practical Physiologic Chemistry, Web- 
ster's Diagnostic Methods and Morris's Clinical Laboratory 
Methods. . 

F. A. F. 

5006 Spruce Street, 
Philadelphia. 



(iii) 



PREFACE TO FIRST EDITION. 



A KNOWLEDGE of SO many branches of medicine is required 
of the medical student of to-day, and as the time at his com- 
mand is comparatively limited, a manual embodying the essen- 
tials of the subject such as this aims to do cannot but prove 
useful. At the same time, it is believed to contain all infor- 
mation necessary to provide a working outline of clinical lab- 
oratory methods for the busy practitioner. 

The book is not intended to take the place of the many 
excellent and exhaustive text-books on Clinical Medicine, but 
rather to supplement them, by pointing out to the busy student 
and practitioner simple and reliable methods by which he may 
obtain the information desired, without unnecessary expendi- 
ture of valuable time upon difficult, tedious or untried methods. 

The author has endeavored in this work to present in as 
concise a manner as possible a selection of the analytical 
methods employed in the clinical laboratory, without burden- 
ing the reader with unnecessary detail, cumbefsome methods, 
etc., many of which are extremely difficult, often requiring 
considerable knowledge of general chemistry and elaborate 
apparatus, which may place them beyond the reach of the 
practitioner. 

For these reasons it is hoped that this little work will 
prove equally valuable to the student and the practicing phy- 
sician. 

In preparing this work special effort has been made to 
bring the subjects treated up to date, by the introduction of 
such new methods as have proven reliable. Some of the 

(V) 



vi PREFACE. 

material is entirely new, and many of the plates and cuts have 
been prepared from original drawings and photographs by the 
author. 

The appendix has been arranged to furnish a working basis 
for the equipment of a clinical laboratory, at the same time 
affording reference for tlie preparation of stains, reagents, etc., 
mentioned in the text. 

The leading authorities have been freely consulted, and 
much material has been obtained from such authorities as 
V. Jaksch, Sahli, Caille, Grawitz, Krehl, Max Braun, Tyson, 
Abbott, Purdy, Eemsen, and Holland. 

The author takes this opportunity to express his appre- 
ciation of the many valuable suggestions received from Drs. 
Judson Daland and Wm. Egbert Robertson, and Mr. Geo. B. 
Johnson, of the F. A. Davis Company; also, to his associate. 
Dr. Francis J. Dever, for invaluable aid in the preparation of 
the manuscript and in correcting the proof. 

F. A. F. 



CONTENTS. 



PAGE 

Section I. The Microscope 1 

Selection of the Instrument, 1. Care of the Microscope, 1. The Parts 
of the Microscope, 2. The Oil-immersion Objective, 5. Apochromatic 
Objectives, 5. Illumination, 6. Dark-ground Illumination, 7. Impro- 
vised Dark-field Illuminator, 8. Cleaning the Microscope, 9. The 
Mechanical Stage, 10. The Warm Stage, 11. The Maltwood Finder, 
11. Examination of Urinary Sediments, 13. Examination of the 
Blood, 15. The Camera Lucida, 16. The Micrometer, 17. 

Section II. The Sputum 19 

General Considerations", 19. Physical and Chemical Characteristics, 
19. Hemoptysis, 24. Macroscopic Examination, 25. Microscopic Ex- 
amination., 26. Stain for Elastic Tissue, 29. Rarer Diagnoses made 
by Examination, 30. Preparation of Stained Specimens, 31. Special 
Methods of Concentrating for Bacterial Examinations, 34. Isolation 
of Tubercle Bacilli, 34. Micrococcus Lanceolatus, 39. Bacillus of 
Influenza, 40. Lepra Bacillus, 41. Bacillus Pertussis, 42. Special 
Reactions, 42. 

Section III. Clinical Hematology 45 

Physical and Chemical Properties, 46. Chemical Composition, 49. 
Methods of Obtaining Specimen, 50. Estimation of Hemoglobin, 51. 
Enumeration of Corpuscles, 56. Cleaning Blood-tubes and Pipettes, 
64. Counting the Blood-platelets, 64. Microscopic Examination, 65. 
Varieties of Leukocytes, 71. Differential Count, 72. Arneth's Classi- 
fication, 75. Leukocytosis, 77. The Anemiias, 78. Chlorosis, 80. 
Leukemia, 81. Hodgkin's Disease (Pseudoleukemia), 83. Degenerated 
Red Cells, etc., 83. Vital Staining, 84. 

Section IV. Spectroscopic Examinations 87 

The Spectroscope, 87. Hemoglobin and its Derivatives, 89. Ives Re- 
plica Diffraction Grating, 92. 

Section V. Newer Methods of Blood Examination 95 

A— Coagulation Time of the Blood, 95. Obtaining the Specimen, 97. 
Methods of Determination, 97. B— Viscosity, 102. Factors Affecting, 
103. Method of McCaskey, 104. Method of Denning and Watson, 106. 
C— Chemical Determinations, 107. Blood Urea, 107. Non-protein Nitro- 
gen, 112. Total Nitrogen, 113. Cholesterol Content of the Blood, 114. 
Alkali Reserve of the Blood Plasma, 118. Carbon Dioxide Tension of 
Alveolar Air, 124. Determinations of Blood Sugar, 129. 

(vii) 



viii CONTENTS. 

PAGE 

Section VI. Sphygmomanometky and Sphygmogeaphy 134 

Capillary Blood-pressure, 134. Arterial Blood-pressure, 134. Sphyg- 
momanometers, 137. Systolic Blood-pressure, 142. Diastolic Blood- 
pressure, 142. Auscultatory Method of Blood-pressure Reading, 143. 
Pulse P*ressure and Mean Pressure, 145. Physiologic Variations in 
Blood-pressure, 146. Pathologic Variations in Blood-pressure, 151. 
Functional Cardiac Tests, 151. Sphygmography, 154. The Polygraph, 
155. Explanation of Normal Pulse Curve, 157. 

Section VIT. Animal Paeasites 159 

Plasmodium of Malaria, 159. Differential Diagnosis of Plasmodia, 161. 
Detection of Plasmodium, 161. Cultivation of Malarial Plasmodium, 
163. Present Status of Parasite of Scarlet Fever, 163. Parasite of 
Yellow Fever, 165. Filariasis, 165. Method of Examination for Filaria, 
167. Sleeping Sickness, 167. Relapsing Fever, 169. Kala-azar, 171. 
Syphilis, 172. Staining Methods, 173. Rapid Staining of Living Spiro- 
chsetes, 175. Animal Parasites, 177. Classification, 178. Protozoa, 179. 
Platyhelminthes, 184. Nematodes, 189. Temporary Parasites, 194. 
Vegetable Parasites, 197. 

Section VIII. Determinations of the Functions of the 

Stomach 200 

The Vomitus, 200. Gastric Contents, 200. Fractional Method of Gas- 
tric Analysis, 201. Method of Rehfus, 201. Preparation of the Patient, 
208. Composition of Test Meals, 209. Daland-Faught Test Meal Ap- 
paratus, 211. Determination of Gastric Contour and Position, 213. 
Chemistry of Digestion, 217. Chemical Composition of Gastric Juice, 
217. Secretion of Hydrochloric Acid, 217. Qualitative Tests, 218. 
Quantitative Tests, 220. Determination of Total Gastric Contents, 221. 
Detection of Organic Acids, 222. Microscopic Examination of Gastric 
Contents, 225. Detection of Leukocytes, 227. Tests for Occult Blood, 
227. Estimation of Peptic Activity, 228. Determination of Pancreatic 
Activity, 232. Determination of Tryptic Activity, 233. Determination 
of Rennin Activity, 234. Conversion of Starch, 235. Stomach Absorp- 
tion Rate, 235. Bile in Gastric Contents, 235. Test for Motor Func- 
tion of Stomach, 236. Indirect Examination of Stomach Contents, 237. 
Rontgen-ray Examination, 238. 

Section IX. The Feces 239 

Physical Characteristics, 239. Study of Intestinal Digestion, 240. 
Determination of Motor Functions of Gastro-intestinal Tract, 241. 
Method for Microscopic Examination, 246. Test for Pancreatic Insuffi- 
ciency, 246. Test for Amylolytic Activity, 247. Chemical Examina- 
tion of Feces, 249. Total Fat, 252. Blood in Stool, 254. Bacteria and 
Protozoa, 260. Clinical Significance of Examinations, 264. Foreign 
Bodies, Calculi and Concretions, 265. 

Section X. The Urine 268 

General Considerations, 268. Decomposition Changes, 269. Preserva- 
tion of Sample, 270. Description and Importance of the Urine, 270. 
Physical Characteristics of the Urine, 271. The Amount, 273. The 
Specific Gravity, 275. Estimating the Total Solids, 279. The Re- 



CONTENTS. ix 

PAGE 

action, 279. Determination of Total Acidity, 281. Folin's Method for 
Acidity, 281. Chemical Composition of the Urine, 282. Inorganic 
Constituents, 282. The Phosphates, 283. The Sulphates, 286. Deter- 
mination of Indican, 287. Skatol, 291. The Chlorids, 292. Purdy's 
Method of Estimating Chlorids, 292. Organic Constituents, 293. Urea, 
294. Estimation of Urea, 299. Uric Acid, 302. Clinical Determination 
of Uric Acid, 304. Purin Bases, 307. Urates, 307. Hippuric Acid, 308. 
Creatinin, 309. Oxalic Acid, 310. Estimation of Total Nitrogen, 311. 
Ammonia, 314. Cystin, 317. Leucin and Tyrosin, 3il8. Abnormal Con- 
stituents, 319. Albumin, 319. Glucose, 327. Volumetric Determination 
of Glucose, 332. Fermentation Saccharimeter, 334. Polarimetric 
Method, 337. Clinical Significance of Glycosuria, 341. Levulosuria, 
341. Lactosuria, 342. Maltosuria, 342. Pentosuria, 342. Acetonuria, 
343. Diacetic Aoid, 345. Oxybutyric Acid, 346. Bile Pigments, 346. 
Bile Acids, 348. Urobilin, 349. Hematuria, 350. Hemoglobinuria, 352. 
Pyuria, 352. Epithelia, 353. Tube Casts, 353. Cylindroids, 357. Casts 
in Relation to Life Insurance Examinations, 358. Inorganic Sediment, 
361. Method to Determine the Nature of Deposits, 364. Urinary Con- 
cretions, 364. Diazo-reaction, 365. Examination for Substances Intro- 
duced from Without, 367. Tests for Renal Capacity, 369. Tests for 
Activity of Other Organs, 376. Dimethylaminobenzaldehyd Reaction, 
377. Hirose Test of Liver Function, 377. 

Section XI. The Body Fluids 378 

Cerebrospinal fluid, 378. Cytologic Studies, 381, Chemical Examina- 
tions, 384. Determination of Protein Content, 384. Oral Secretions, 
387. Nasal Secretions, 391. Conjunctival Secretions, 391, Transudates 
and Exudates, 391, Physical and Chemical Properties, 393. Varieties 
of Exudates, 395. Microscopic Examination, 396. Peritoneal Fluid, 
397. Cyst Fluids, 398. Pleural Fluid, 399. Pericardial Fluid, 401. 
Synovial Fluid, 401. Hydrocele Fluid, 401. 

Section XII. Human Milk , , , , 402 

General Consiiderations, 402, Physical Characteristics, 402. Micro- 
scopic Ex;amination of, 403, Fat Determination, 404, Sugar Deter- 
mination, 405, Protein Determination, 405. Tests for Formalde- 
hyde, 406. 

Section XIII. Bactekiologic Methods 407 

Sterilization, 407. Sterilization by Heat, 408. Chemical Sterilization 
and Disinfection, 415. Preparation of Culture Media, 416. Preparation 
of Tubes, etc. for Culture Media, 422. Technic for Plates ajid Petri 
Dishes, 423, The Incubator, 424. Common Disease-producing Organ- 
isms, 426. Tuberculosis, 426, Bacillus of Diphtheria, 428. Gono- 
coccus of Neisser, 429. Meningococcus, 430, Typhoid Bacillus, 430. 
Classification of Bacteria, 432. LoflSer's Method, 434. Capsule Stain- 
ing, 436. 

Section XIV. Sebodiagnosis 438 

Agglutination, 438, Specific Typhoid Reaction, 438, Agglutination Re- 
actions in Other Diseases, 441. Mendelbaum's Test for Typhoid, 443. 
Principles of Wassermann and Noguchi Reactions, 445. Leutin Re- 
action, 460. Meiostagmin Reaction, 463. 



X CONTENTS. 

PAGE 

Appendix 470 

Clinical Terms, 470. Diseases in which Laboratory Tests are of 
Value, 470. Urinalysis Report Form, 476. Method of Fixing Blood 
Smears and Films, 477. Diluting Solution for Blood Cell Count, 477. 
Diluting Fluid for Blood Platelet Count, 478. Blood and Bacterial 
Stains, 479. Labelling Smears, 488. Substances to Prevent Foaming, 
488. Normal and Decinormal Solutions, 489. Standard Solutions, 493. 
Special Reagents and Solutions, 494. Indicators, 498. Tables, 505. 
International Atomic Weights, 505. Metric Weights and Measures, 
505. Metric Equivalent of Multiples in a Grain, 506. Metric Equiva- 
lent of Multiples of a Minim, 506. American Equivalent of Metric 
Fluid Measures, 507. American Equivalent of Metric Measures of 
Mass, 507. Comparison of Thermometric Scales, 508. Bang's Table of 
Copper Reduction Equivalents, 509. Blood-pressure Chart, 510. Table 
for Converting Apothecaries Weights and Measures into Grams, 511. 

Index 513 



LIST OF ILLUSTRATIONS. 



PAGE 

Plate I. Tubercle Bacilli in Sputum. (Colored) 32 

Plate II. Normal and Pathological Blood-cells. (Colored) 68 

Plate III. Absorption Spectra of Hemoglobin and its Derivatives 9(i 

Plate IV. Sphygmomanometer in Position for Observation ISC. 

Plate V. Malarial Parasites. (Kolle & Wassermann) ]60 

Plate VI. Intestinal Parasites of Man 180 

Plate VII. Indican Scale. (Colored) , 288 

Plate VIII. Uric Aoid Crystals with Amorphous Urates. (Peyer.) (Colored) 308 

Plate IX. Uric Acid Crystals 363 

Plate X. Calcium Oxalate Crystals and Phosphates 363 

Plate XI. Uric Acid Crystals, Cholesterin, Cystin, Tyrosin and Leucin . . 363 

FIG. 

1. Microscope 2 

2. Dark-field Illuminator 7 

3. Metallic Stop for Leitz Microscope 8 

4. Mechanical Stage , 10 

5. Maltwood Finder 12 

6. Camera Lucida 16 

7. Bronchial Cast, from Case of Fibrinous Bronchitis in Service of Dr. 

Judson Daland (Original) 22 

8. Actinomyces showing Radial Formation 30 

9. Pycnometer 47 

10. Fleischl Hemoglobinometer 51 

11. Fleischl-Meischer Hemoglobinometer 53 

12. Sahli's Hemoglobinometer 54 

13. Dare's Hemoglobinometer 55 

14. Levy-Neubauer Hemocytometer 57 

15. Counting Chamber with Single Neubauer Ruling 58 

16. Daland Hematokrit. (A. H. T. Co.) 59 

17. Detail of Neubauer Ruling 63 

18. A Computing Chart for Differential Leukocyte Estimation. (Devised by 

Dr. A. E. Osmond) 73 

19. Spectroscope 87 

20. Direct Vision Spectroscope 88 

21. Set-up of Ives Grating 92 

22. Measurement of Wave Length 93 

23. Boggs's Coagulometer. (A. H, T. Co.) 98 

24. Dorrance's Coagulometer 99 

25. Test-tubes 108 

26. Folin Ammonia Absorption Tube 10« 

27. Van Slyke and Cullen Urea Apparatus HO 

28. Kjeldahl Flasks 113 

29. Cholesterol In Blood-method of Myers Apparatus 115 

30. Ph Concentration 119 

30a. Hydrogen-ion Concentration Colorimeter 121 

31. Marriott Alveolar Air Testing Outfit 125 

32. Epstein Apparatus for Blood Sugar 129 



(xi) 



xii \y' LIST OF illust:^ations. 

FIG. PAGE 

33. Normal Pulse Tracing: Showing Relation of Systolic, Diastolic, Pulse- 

pressure, and Mean. Pulse-pressure equals 30 135 

34. Faught's Mercury Sphymomanometer 137 

35. Faught's Clinical Sphygmomanometer 138 

36. Actual Size Pocket Indicator 139 

.37. Enlarged Diagram of the Author's Pocket Indicator 140 

38. Stamp Bracelet in Use 146 

39. Woley's Chart Showing Effect of Age on Blood-pressure 147 

40. Dudgeon's Sphygmograph 155 

41. Jaquet's Polygraph 366 

42. Sarcoptes Scabiei 192 

43. Pediculus Pubis 156 

44. Achorion Schonleini , 198 

45. Trichophyton Spores and Threads 199 

46. Normal and Pathologic Curves 201 

47. Rehf uss Tube and Extraction Syringe 202 

48. Acidity Curves of Normal Human Stomach 203 

49. Acidity Curves from Case of Hyperacidity 204 

50. Acidity and Protein Curves in Gastric Carcinoma 205 

51. Total Acidity and Protein Curves in Benign Achylia 206 

52. Author's Apparatus for Gastric Test-meal Removal, Lavage and Inflation. 207 

53. Diagrammatic Representation of Arrangement of Bottles for Measuring 

Cubic Contents of Stomach 215 

54. Strauss's Separatory Funnel 223 

55. Boas-Oppler Bacillus in Gastric Contents 226 

56. Strasburger Apparatus. (After Steele) 251 

57. Urinometer and Cylinder 277 

58. Urinometer 303 

59. Ruhemann's Uricometer 305 

60. Urate of Soda and Crystals of Uric Acid, Oxalate of Lime, and Cystin . . 308 

61. Folin's Ammonia Apparatus 316 

62. Improved Esbach Albuminometer 324 

63. Einhom Saccharimeter in Use 335 

64. Ultzmann's Polariscope 339 

65. "Large White Kidney." X350 354 

66. Chronic Bright's Disease. X350 354 

67. Sedimentation Glasses 355 

68. Centrifuge Tubes 362 

69. Duboscq Colorimeter 371 

70. Hellige or Universal Colorimeter 372 

71. Dunning Colorimeter 373 

72. Kober Colorimeter 374 

73. Organisms of Vincent's Angina 390 

74. Arnold Steam Sterilizer. (A. H. T. Co.) 413 

75. Autoclave. (A. H. T. Co.) 414 

76. Thermostat or Incubator. (A. H. T. Co.) 425 

77. Portable Clinical Laboratory Set 469 

78. A Graphic Chart for Recording Blood-pressure Observations 510 



I. 

THE MICROSCOPE. 
ITS ACCESSORIES AND MICROSCOPIC TECHNIC. 



SELECTION OF THE INSTRUMENT. 

In purchasing a microscope one should consider nothing 
but the best. This is true economy, for a cheap or inferior in- 
strument is always an unsatisfactory one, which, when once 
bought, is hard to get rid of, and if later discarded for a better 
instrument, represents a total loss. The possession of a good 
microscope is an absolute essential. Its services are required in 
almost every investigation of modern clinical diagnosis, and 
there is hardly a chapter in this work which does not call for 
its use. While the finest and most expensive instruments are 
still of foreign make, there are, nevertheless, many of American 
make which compare favorably with the imported instruments. 
An example of a good American instrument is shown in Fig. 1, 
and is one of a series manufactured by the Bausch and Lomb 
Optical Co. 

CARE OF THE MICROSCOPE. 

In handling any instrument it is necessary to see that it 
is always grasped by some one of its solid parts — the base or 
the standard. Some of the newer makes are provided with an 
aperture in the standard for this very purpose. 

If the instrument is to be in daily use it should be kept 
under a bell jar or in a specially prepared cabinet built above 
the work table. Eeference to Fig. 1 will illustrate the mechan- 
ism and the different parts with which one should become 
familiar before attempting to use the instrument. Efforts to 
use the microscope by any one unfamiliar with its different parts 
and adjustments should never be permitted, since careless 

(1) 



2 THE MICROSCOPE. 

handling ma}^ result in serious damage to the objective or other 
delicate parts. 

DESCRIPTION OF THE MICROSCOPE. 

Referring to Fig. 1, the various parts of the instrtiment 
about to be described ma}^ be located. 



OCULAR 
DRAW TUBE 



COARSE 

ADJUSTMENT 



FINE 
ADJUSTMENT 




TUBE 



TRIPLE 
NOSEPIECE 
V^ITH OB- 
JECTIVES 



—STAGE 



IRIS 
DIAPHRAGM 



MIRROR 



Fio. 1.— Microscope. 



The Ocular, or Eye-Piece, consists of one or more converg- 
ing lenses, the combined action of which is to magnify the 
image formed by the objective. This part is contained in a 
tube of its own, which telescopes into the top of the barrel, and 
is the part which the eye approaches when viewing the object. 



DESCRIPTION OF THE MICROSCOPE. 3 

Various oculars are usually provided, and are numbered 
from 1 to 8. As the number advances, the magnifying power 
of the ocular increases; at the same time the length of the 
tube decreases. It is advised, when increased magnification is 
desired, to accomplish this by increasing the power of the ob- 
jective rather than by using stronger oculars. The clearness 
and definition of the picture will then be preserved, whereas 
if the power of the ocular is increased it is usually at the 
expense of definition. 

It will be found of great convenience to cement into one 
of the oculars a fine hair or eyelash. This should rest upon 
the upper surface of the central diaphragm of the ocular, when it 
will always be in focus. This serves as a pointer which can be 
used to single out from a blood or other specimen some special 
point of interest that it is desired to demonstrate to another 
person. This is particularly valuable in class demonstration. 

The Barrel, or Tube, is the large tube of the microscope, 
which serves as a conductor of the rays as they pass from the 
objective to the ocular. 

The Objective consists of a system of converging lenses 
arranged at the lower end of the barrel, where it forms a mag- 
nified inverted image of the object. Upon this piece depends 
the magnifying power of the microscope. Most objectives are 
designated by numbers running from 1 to 15, these numbers 
representing the fractions of an inch at which the lens operates. 
Foreign makes are designated by letters which are not in any 
way directly comparable to the numbers of the American objec- 
tives. For one desiring three objectives, the most useful num- 
bers will be found to be the 3, the 7 or 6, and the 12-oil-im- 
mersion, the 3 and 6 or 7 being chiefly employed in blood-count- 
ing, while the 12th is necessary for blood-examination and bac- 
teriologic work. 

The Stage is the table fastened below the barrel and in a 
right-angle plane to it. This serves to retain the object in a 
horizontal plane to the optical axis of the instrument. The 
table is provided with spring clips to better hold the slide in posi- 
tion during examination. The newest Leitz scope is furnished 
with a movable (not mechanical) stage, which, by the operation 
of two milled screws, is capable of slight movement in all direc- 



4 THE MICROSCOPE. 

tions. This^ when a mechanical stage is not at hand, is of great 
service in searching blood and bacteriologic slides. 

The Reflector is a small mirror situated below the stage 
which serves to direct the ra3^s of light upward through the 
object in the optical axis of the microscope. The reflector has 
two sideSj one carrying a concave, and the other a plane, mirror. 

The Sub-Stage Condenser (Abbe's) consists of a system of 
lenses arranged between the stage and the reflector. The object 
of this part of the instrument is to collect and condense the 
rays as they come from the reflector, so that they are focused 
upon the object, thus furnishing brilliant illumination. 

The Iris Diaphragm is now universally employed for con- 
trolling the intensity of the illumination. This is held in the 
same carrier as the condenser, and is just below it. By means 
of a lever every gradation of light, from the most intense to 
absolute darkness, is readily obtained. The proper manipula- 
tion of this diaphragm constitutes a very important part of the 
practical knowledge gained from the use of the microscope, and 
has much to do with the success of many investigations. 

The Adjustments. — The coarse adjustment is the rack-and- 
pinion mechanism projecting from the upper part of the stand- 
ard, and is employed to rapidly raise and lower the barrel and 
its attachments. The fine adjustment is a micrometer screw 
situated usually below the rack and pinion. This serves the 
purpose of very gradually raising or lowering the barrel in order 
to obtain exact focus. 

The Draw-Tube is a very important adjunct of high-grade 
instruments, because by its skilful manipulation slight errors 
in refraction, due to inequalities in slide or cover-glass, may be 
corrected. 

The Nose-Piece, or collar, is fastened to the lower end of 
the barrel, and permits the attachment of two or three objectives 
at one time in such a position that, by rotation of the collar, 
any one of them may be immediately brought into the axis of 
the instrument. 

The most important accessories of the microscope are the 
objectives, and the quality of all microscope work largely de- 
pends upon their perfection. While the quality of objectives 
vary much, one cannot go far astray if they are obtained through 



APOCHKOMATIC OBJECTIVES. 5 

a reputable supply house or from a well-known manufacturer, 
such as the Zeiss or Leitz abroad, and the Bausch and Lomb 
Optical Co., U. S. A. 

THE OIL-IMMERSION OBJECTIVE. 

The oil-immersion objective, or homogeneous system, is so 
constructed that when in use the pencil of light passing through 
the object to the objective traverses only media of the same 
refractive index. This is accomplished by placing between the 
cover-glass and the end of the objective a medium having the 
same refractive index as glass. To accomplish this a drop of 
cedar oil is placed upon the cover-glass, and the objective brought 
into contact with this, and the observation made through the oil. 
This class of lens is intended to work only with the oil, and is 
unsatisfactory when used dry. Frequently it is not convenient 
to use cedar oil for the preliminary examination often employed 
to determine the progress of staining, etc. ; here the staining 
fluid may be washed off and a drop of water used in place of 
the oil; this will give a sufficiently good picture for the pur- 
pose, and has the advantage of not interfering with the addition 
of further stain when desired. 

The tube or barrel of the microscope is made in two forms, 
the long or English type being from 8 to 10 inches, and the 
short or Continental type having a length of G^/o inches. The 
latter is the more desirable form, since the majority of manu- 
facturers of objectives adjust them for the short tube. 

APOCHROMATIC OBJECTIVES. 

This term is applied to a particular variety of lens contain- 
ing a special kind of glass (containing calcium fluoride), be- 
sides the usual crown and flint glass, the object of this being 
to produce a greater degree of achromatism, thus reducing 
chromatic aberration. The special value of these objectives 
when used with a compensating ocular (compensating eye-pieces 
are specially constructed for use with apochromatic objectives), 
is as follows: Three rays of tlie colored spectrum, instead of 
two, as in case of the achromatic glasses, are focused in the 
same plane, leaving only a minute tertiary spectrum : also with 
these objectives the spherical aberration is corrected for two 



6 THE MICROSCOPE. 

colors in the brightest part of the spectrum, and the objective 
shows the same correction for marginal rays as for the central 
part of the aperture. Finally, apochromatic objectives admit 
the use of highly magnifying oculars. This form of objective is 
particularly useful in photomicrographic work, where it is very 
important to abolish chromatic aberration. 



ILLUMINATION. 

Illumination constitutes a most important factor in the 
practical use of the microscope, and upon its proper manage- 
ment depends very much of the efficiency of the work. Direct, 
unmodified sunlight is unsuited, and should not be employed, 
for general work. North light is the most uniform and steady, 
and is to be preferred. The use of artificial light should, as far 
as possible, be avoided, and when it must be used its character 
and color may be greatly im^proved by inserting a piece of blue 
glass between the reflector and the object. It is always neces- 
sary to keep the eye close to the ocular and if possible to keep 
the unemployed eye open. Too much light is always to be 
avoided, as it is only an added strain to the eyes, accomplishes 
nothing, and may materially interfere with the efficiency of the 
work. 

Oblique Light. — For urinary work the oblique light is best. 
By this term is meant light in which the parallel rays from the 
plane mirror meet the optical axis of the microscope at an angle. 
This form of light may be obtained in the following ways : (a) 
By placing the reflector to one side of the stage; this results 
in an oblique ray without materially lessening the strength of 
the light; the condenser must, of course, be swung out of the 
way. In microscopes Avhich have a fixed mirror, i.e., only mov- 
able perpendicularly at right angles to the axis, oblique light 
may be obtained through the condenser, as follows: (h) First, 
focus the light upon the object through the condenser; then, 
lower the condenser until its focus is considerably below the 
plane of the object; thus the rays emerging from the condenser 
will decussate and then diverge, so that all but axial rays will 
fall upon the object in an oblique direction. 



DARK-GROUND ILLUMINATION. 7 

DARK-GROUND ILLUMINATION. 

The Substage Dark-ground Illuminator. — Most high-grade 
microscopes are now fitted with a sub stage dark-ground illumi- 
nator. (See Fig. 2.) If this, is available, the microscope should 
be set in a vertical position and the dark-ground illuminator sub- 
stituted for the regular Abbe. If daylight is used, no paralleling 
device is necessary. If artificial light is used a bulls-eye con- 
denser must be interposed between the substage reflector and 
the light, in such a way that parallel rays strike and completely 



AlA 





Fig. 2.— 'A, Dark-ground Illuminator; B, Sectional View of Dark-ground 
ILLUMINATOR— Showing Path of Ray of Light. 



cover the plain substage mirror. Then focus the two concentric 
riQgs engraved upon the upper surface of the condenser and 
center them accurately by means of the screws provided for this 
purpose. 

Preparation of Fresh Specimen. — When employing the dark- 
field condenser the material to be examined should be in a fresh, 
moist state, if the best results are to be obtained. Carefully 
smear the specimen to be examined upon one or more slides, 
preferably new ones that have not been scratched by handling, 
and cover with thoroughly cleansed new No. 1 cover-glasses. 
Place a large drop of immersion-oil upon the upper surface of 
the condenser and then fasten the specimen on the microscope 
stage, bringing the condenser up until the immersion-oil is in 
contact with the under surface of the slide. A properly adjusted 
mirror should show a bright spot in the center of the mounted 



8 THE MICROSCOPE. 

slide. Place a drop of cedar oil in the center of the cover-glass 
and bring the intermediate objective into contact with the drop 
of immersion-oil; focus the bright spot already referred to, and 
if it does not occupy the center of the field adjust the substage 
mirror until it does. If these steps are properly carried out, 
and the object to be examined is properly in focus, then the 
intensely illuminated bacteria, spiroch£ete, etc., will stand out 
sharply against a dark or black background. 

IMPROVISED DARK-FIELD ILLUMINATOR. 

For one not possessing a dark-field illuminator, a very satis- 
factory substitute can be made by having a piece of thin metal 




Fig. 3.— Form and Size of Metallic Stop, Furnished with the 

Leitz Microscope, Used in Conjunction with the Abbe 

Condenser, to Produce dark-field illumination. 

cut in the form shown in the illustration (Fig. 2) of such a size 
that the outer narrow ring fits snugly into the movable ring pro- 
vided in most substage Abbe condensers. This special stop is 
now furnished with most microscopes. A little experience 
will indicate the proper size of the central disc, which when 
properly adjusted should produce conditions similar to those 
described in the preceding paragraph. The relation of con- 
denser, specimen, and objective is the same as with the regular 
dark-field illuminator. Eesults with this apparatus, while not 
perfect, are very satisfactory and may be relied upon for demon- 
strating spirochsete and other micro-organisms. It has the ad- 
vantage of allowing the examiner to rapidly shift from the dark 
to the light field without disturbing the relation of the objec- 
tive, specimen or condenser. 



TO CLEAN THE MICROSCOPE. 9 

TO CLEAN THE MICROSCOPE. 

Should the lenses or oculars become blurred or spotted from 
dust or dirt, proceed as follows : If the dirt is upon the ocular 
it will be discovered by rotating the ocular within the barrel 
while observing the illuminated field of the microscope. If the 
obstniction is upon the ocular it will be seen to move; if upon 
the objective it will remain stationary during this manipula- 
tion. To find an obstruction upon the objective rotate it, when 
the spot or mark will move with it. Finally, if after testing 
both the objective and ocular in the manner described above the 
location of the dirt or dust is not demonstrated, the trouble will 
then be found upon the glasses or spectacles if the observer 
happens to wear them. Having located the trouble, remove the 
affected part and cleanse as follows : If careful polishing with 
bibulous paper or fat-free silk fails to accomplish the desired 
result, then moisten the silk or paper with a trace of distilled 
water, using this to aid in the removal, finally polishing dry. 
Tor oily or resinous smears a small amount of alcohol may be 
used. Special care should then be exercised to avoid any excess 
of the solvent which may enter between the individual lenses 
and, dissolving the cement, ruin the part. After using the oil- 
immersion it should always be cleaned before leaving the instru- 
ment. After removing the excess of oil with the silk, wipe off 
the remainder with a trace of xylol or benzine, immediately 
wiping dry. Cover-glass preparations may be treated in a 
similar way. 

Grlass surfaces should never be touched with the fingers, 
and great care should be exercised to avoid dropping and possi- 
ble fracture of the oculars and objective. All metal parts of 
the instrument should be kept free from liquids, particularly 
acids and alkalies, benzine, xylol, alcohol, turpentine, and 
chloroform. 

To clean the mechanical parts, none but the best machine 
oil should be used, and this only in small amounts and at long 
intervals. The polished brass requires nothing but occasion- 
ally polishing with clean chamois. 

Two keynotes to successful use and preservation of the 
microscope are: handle with care and keep scrupulously clean. 



10 



THE MICROSCOPE. 



THE MECHANICAL STAGE. 

Of the many aids to exact work in the realm of clinical 
medicine the mechanical stage is of great practicability and 
wide application. Every possessor of a microscope should aspire 
to the possession of a mechanical stage, for this mechanism is 
not only a great time saver, but will materially aid in the search 
for bacteria and pathologic cells in the blood, and is practically 
a necessity in making a differential count. 

Bescription. — Eeferring to Fig. 4, the general appearance 
of this instrument will be seen. It is designed for application 
to the stage of the microscope, upon which it is rigidly fastened 




Fig. 4.— Mechanical Stage. 

by a collar and set screw attached to the stand. It is important 
in this connection to note that most manufacturers of micro- 
scopes make a mechanical stage for their particular instrument, 
and which frequently is not interchangeable with microscopes 
of other manufacture, so that, before purchasing, one should be 
certain that the stage will fit the instrument for which it is 
intended. When attached to the stand the slide carrier should 
move in both directions without any undue force and without 
any irregularity or jarring of the mechanism, and to the limits 
of its movement should remain in close and even contact with 
the microscope stage. 

The mechanical stage is fitted with two milled screws 
which are movable in either direction, and which convey gradual 



: THE MALT WOOD FINDER. H 

motion to the slide held in the jaws of the instrument. One 
of these screws controls the vertical, and the other the hori- 
zontal, motion, so that by proper manipulation it is possible 
to rapidly and accurately go over the whole of a specimen. (For 
more detailed description of the use of the mechanical stage in 
the differential count, see chapter on ^^The Blood.") 

Most mechanical stages are provided with a millimeter 
scale and vernier reading to 0.1 of a millimeter, which serves 
not only to measure the size of small objects upon the stage, 
but is also very useful for locating objects of special interest 
upon any slide, so that they may again be found without dif- 
ficulty. (See below the Maltwood finder and Pepper's applica- 
tion without the mechanical stage.) 

THE WARM STAGE. 

A further differentiation and improvement upon the me- 
chanical stage is the stage prepared for preserving specimens at 
body-temperature during examination. This is provided with a 
thermometer which indicates the temperature of the object 
during examination. An extemporaneous warm stage has been 
described in another section (Parasites) to which the reader is 
referred. 

THE MALTWOOD FINDER. 

In microscopic work, especially in studying blood or bac- 
teriologic slides, some sort of "finder" is an essential part of the 
equipm^ent. While the vernier scale, attached to most mechanical 
stages, is fairly satisfactory for individual work, it does not 
fulfill all conditions demanded of it, since the same stage and 
microscope must always be used, and if by accident the relation 
between microscope and stage is altered ever so little, then all 
previous figures indicating location are rendered valueless. 

The Maltwood finder (Fig. 5) does not possess the above 
disadvantages, and can be used universally with uniform results. 

The M'altwood finder^ consists of a heavy glass slide com- 
paring exactly in size with the ordinary microscope slide. The 
central third of this slide is covered with a close network of 



1 W^m. Pepper: Jour. Amer. Med. Assoc, July 20, 1908. 



12 



THE MICROSCOPE. 



intersecting rectilinear lines which form a large number oi 
uniform squares. Each square contains two figures arranged 
one above the other^ so that no two squares represent the same 
combination (see below). This marking has been placed upon 
the slide by a photographic process. 



1 


1 


1 


1 


1 


1 


2 


3 


4 


5 


2 


2 


2 


2 


2 


1 


2 


3 


4 


5 


3 


3 


3 


3 


3 


1 


2 


3 


4 


5 



All Maltwood finders are made interchangeable, the squares 
coinciding exactly in all slides. 

Method of Using the Finder. — If on looking over the slide 
with the mechanical stage a part of the field is discovered which 







Fig. 5. — Maltwood Finder, showing Dr. Pepper's Brass Angle 
Substitute for Mechanical Stage. 

one desires to examine at a later time, the slide is carefully re- 
moved without altering the location of the mechanical stage 
and the Maltwood finder substituted; the square is then ob- 
served which corresponds with the center of the slide. This 
combination is to be jotted down, and then in order to make 
sure that the proper reading has been made, the finder and slide 
should be substituted for each other a number of times. Sup- 



EXAMINATION OF URINARY SEDIMENTS. 13 

pose this reading was made ^^ , then, when later this par- 
ticular field is sought the above process is reversed, and when 
the recorded square is in the center of the field, the slide is care- 
fully substituted, when the object sought will appear in the field. 

One of the chief advantages of the finder is that the slide 
may be sent to any one having a finder, with a note to look for 
such or such a square. For those not possessing a mechanical 
stage, Dr. W. Pepper has devised a small right angle of brass 
which may be easily carried in the pocket, and which may at 
any time be substituted for the mechanical stage, as the only 
purpose of the mechanical stage is to afford a fixed angle into 
which the slide and finder may be fitted. 

The illustration shows the arrangement of slide and brass 
angle, and illustrates the size of the right angle compared to 
the slide. The strip of brass from which the form is cut should 
be about one-sixteenth inch thick. 

EXAMINATION OF URINARY SEDIMENTS. 

Preparation of the Slide. — After concentration of the sedi- 
ment by centrifugation or sedimentation, the next point is to 
arrange this collection upon the slide for examination under 
the microscope. For this examination a few drops may be taken 
up in a pipette and allowed to fall upon the center of a clean 
slide, upon which a clean cover-glass is immediately placed. 
This will exclude dust, prevent rapid drying during examination, 
and will so flatten the field that frequent change in focus will 
not be necessary, while the different elements are more easily 
viewed and differentiated owing to their uniform separation and 
lack of overlapping. 

The proper removal of the sediment from the bottom of the 
container is an all-important part of this process, and upon 
this the successful finding of casts will frequently depend. This 
is best accomplished with a small nipple-pipette or section of 
narrow glass-tubing, one end of which is drawn out into a coarse 
capillary point. Having cleansed the slide and cover slip, ar- 
range them in a convenient place and then proceed to remove 
a few drops of sediment from the bottom of the tube. It is 
absolutely necessary, during this procedure, to prevent the en- 
trance of an excess of fluid into the pipette. This end is accom- 



14 THE MICROSCOPE. 

plished in the case of the pipette by carrying it down into the 
fluid nearly to the sediment, then expressing a few bubbles of 
air before passing the tip beneath the sediment. With the 
straight tube the moist thumb or finger is held firmly upon the 
upper opening of the tube while its tip is carried to the bottom 
of the vessel, then by means of a slight rotation of the tube 
while relaxing the pressure above, a few drops of ^sediment are 
allowed to enter the tube. Firm closure of the upper end of 
the pipette is maintained while the tube is withdrawn and its 
contents transferred to the slide. To further guard against 
diluting the sediment, the outside of the tube should be wiped 
dry after it has been removed from the urine. 

Special Technic for Excessive Sediments of Phosphates, 
Pus, etc. — It is not uncommon to meet with urinary sediments 
of such volume and density that the grosser condition may com- 
pletely obscure the more important but less numerous elements, 
such as casts, pus, blood-cells, etc. In such cases the following 
technic is advised: After taking up the sediment deposit but 
one drop upon the slide; then from a clean pipette add a few 
drops of distilled water or, what is better, the clear urine from 
above the sediment. Agitate this till evenly mixed, and then 
apply the cover-glass and remove excess of fluid with strips of 
filter- or blotting-paper. Thus diluted not only is there better 
opportunity of finding casts, but the internal structure of the 
different elements will be much clearer. 

Microscopic Search. — After preparation of the specimen as 
above outlined, the slide is transferred to the horizontal stage 
and first viewed by low power and a rather subdued light. In 
this examination it is safer to begin with the objective below 
the focal point, then with the eye in position gradually rack 
upward, at the same time keeping the slide moving with the 
other hand. If this is continued carefully there will be little 
difficulty in discovering the objects in the sediment, even if few 
and small. Another advantage of this plan is that all danger 
of crushing the slide or fracturing the objective by forcibly 
racking down is eliminated, since the objective is never carried 
downward except when the relation of objective to slide is plainly 
seen. 

Having focused the sediment and regulated the light, the 



EXAMINATION OF THE BLOOD. 15 

slide should be searched as follows: Slowly move the slide for- 
ward until one corner of the cover-glass appears in the field, 
then locate the lower right-hand corner and gradually move the 
slide upward in as straight a line as possible, until the opposite 
border is reached. Then move slightly to the left and proceed 
in a straight line downward to the lower margin again. This 
procedure is repeated until the whole area of the cover-glass 
has been searched. For those having a mechanical stage the 
slide may be placed in this, and the same movements carried 
out with its aid. In either case it is well to keep one hand upon 
the fine adjustment to sharpen up any obscure objects which 
may be encountered. 

EXAMINATION OF THE BLOOD. 

For an examination of fresh blood the 6 objective is most 
suited, since nothing but the cell outlines can be seen with the 3. 
A good light, with the condenser in close apposition with the 
under surface of the slide, will give the best results. To differ- 
entiate the varieties of white cells by their nuclei and granula- 
tion requires very careful regulation of the light, since the very 
slight difference in refraction of nuclei and protoplasm makes 
the differentiation extremely difficult at best. 

Counting the Corpuscles: Bed Cells. — Having made the 
proper dilution and mounted the specimen as outlined in the 
section on blood, the slide is placed upon the stage and a few 
minutes allowed to pass while the corpuscles settle to the bot- 
tom of the chamber. The medium power, 6, is brought into 
close apposition with the cover-glass, and then with the eye to 
the ocular the fine adjustment will bring the individual cells 
into view. Considerable difficulty is at times experienced in 
locating the part of the counting chamber containing the ruled 
lines. This may be overcome by centering the inner circle of 
the chamber by means of the outside of the objective. If this 
is carefully done the first attempt at focussing will usually bring 
the squares into view. Some hemocytometer slides are very 
faintly ruled, rendering the operation of counting very difficult. 
This may, in a measure, be overcome by reducing the light, at 
the same time taking advantage of the slight shadow caused by 
oblique rays coming from the lowered condenser. If this is done 



16 THE MICROSCOPE. 

great care should be observed in determining border-line cells on 
account of the uncertain shadow cast by all cells with this 
illumination. 

The White Cells. — Either the 3 or the 6 objective may be 
used in this connection, and of these the 3 is generally pre- 
ferred. It does, however, require a little more care and experi- 
ence to make an accurate count, because of the minute appear- 
ance of the individual cells. Its chief advantage is that it is 
not necessary to move the slide while counting the entire field, 
after it has once been centered, thus doing away with the error 
of missed squares which occasionally occurs in moving the 
slide when using the 6 objective. Here, again, low illumination 
and oblique rays may advantageously be employed. 



Fig. 6.— Camera Lucida, When in Use. This Attachment 
is clamped to the ocdlar. 



Examination of the Stained Specimen. — Since the usual 
reason for staining and examining the dried specimen is to 
study minute structural characteristics, to make a differential 
count ;-or to discover the presence of bacteria or parasites, it is 
necessary to employ the oil-immersion lens and to occasionally 
supplement this with a highly magnifying ocular. In this work 
good illumination and sharp definition (condenser close to 
slide) are essential. 

THE CAMERA LUCIDA. 

The CAMERA LUCIDA is a device by the aid of which it is 
possible to quickly and accurately trace upon paper the magnified 
image of any microscopic object upon the microscope stage, 
It will be found of particular value in parasitology, where it is 
required to study in detail the minute anatomy of organisms too 
large to be viewed at one time under the microscope. 



THE MICROMETER. 17 

This apparatus is essentially a combination of mirrors and 
lenses by which the image on the sheet of paper is reduced by 
a suitable lens and is projected into the field of the microscope, 
so that the eye of the observer at the eye-piece of the camera 
lucida sees the image of the object and the paper and pencil of 
the examiner at the same time, i.e., apparently the enlarged 
object has been transferred to the sheet of paper, so that the 
pencil, as followed by the eye, can be made to trace the lines of 
the object upon the paper. 

The essential parts of the apparatus are the eye-piece, which 
is arranged to clamp firmly above the ocular of the microscope 
in the optical axis of the instrument. This contains mirrors and 
lenses suitably arranged to reduce and deflect the image pro- 
jected from the large reflecting mirror situated to the right of 
the ocular. This large mirror serves to reflect the image of the 
paper and pencil tip into the condensing lens of the camera 
lucida. 

In setting up this instrument, the circular clamp on the 
camera lucida is firmly attached to the upper part of the ocular, 
and the horizontal bar carrying the reflecting mirror is clamped 
into the guide provided for that purpose. The camera lucida 
is so hinged to the clamp that it can be swung out of the way 
while locating the proper field in the microscope, after which it 
can be swung into place and centered by means of two small 
setscrews. The reflecting mirror is also arranged so that its 
angle can be adjusted between 45 and 80 degrees. 

THE MICROMETER. 

This is an instrument used for measuring the size of bac- 
teria or other objects, when viewed through a microscope. 

The unit of length employed in micrometry is the one- 
thousandth part of a millimeter (0.001 mm.), called for short a 
micron and indicated by the Greek letter P. 

The first requirement is a stage micrometer; this is a slide 
having engraved on it a scale divided into hundredths of a milli- 
meter (0.01 mm.), every tenth line being longer than the others 
to facilitate counting. The face of this scale is protected by a 
cover-glass, which is cemented over it. 

2 



18 THE MICROSCOPE. 

The measuring is done with a camera lucida in conjunction 
with the stage micrometer as follows : — 

Adjust the camera lucida to the eye-piece of the microscope, 
then adjust the micrometer on the stage of the microscope and 
accurately focus the division. Project the scale upon a piece 
of paper and with a pen draw accurately the magnified image 
of the scale. This is preserved for future use. In using this 
paper scale, the same combination of ocular, objective, and tube- 
length should always be maintained. A similar scale to suit all 
combinations may be made in a like manner. 

Eye-piece Micrometer. — This is a circular glass disc having 
engraved on it a scale divided into tenths of a millimeter (0.1 
mm.). This is fitted inside the ocular in such a way that it 
rests upon the central diaphragm. When in this position it is in 
most instruments exactly in focus of the eye-lens. 

This eye-piece micrometer must then have the value of its 
spaces, according to each optical combination, estimated with the 
stage micrometer and the measure of the spaces together with the 
optical combination should be recorded for future use. 

In measuring an object by this method, read off the number 
of divisions of the eye-piece micrometer which it covers, and 
express the result in microns by reference to the corrected table 
previously prepared. 



11. 

THE SPUTUM. 



GENERAL CONSIDERATIONS. 

Sputum^ or expectoration, is the product of inflammatory 
reaction of the bronchial and lung tissue and is voided by cough- 
ing or clearing the throat. It is composed of the secretion and 
the exudate from the mucous membrane of the nose, pharynx, 
and trachea, down to the finest bronchioles and alveoli; also 
material that may have entered the respiratory tract from ad- 
jacent organs (pus of abscesses and empyema) ; blood derived 
from any part of the respiratory tract; and of material coming 
from the buccal cavity and from any part of the digestive tract. 

On account of this complex origin the composition of the 
sputum is very variable. The sputum may be present, but may 
not be expectorated. Small children and occasionally adults, 
on account of bad habits, insufiicient practice, or impaired con- 
sciousness, swallow their sputum. In the majority of cases this 
difficulty can be remedied by training. For diagnostic purposes 
the total output of sputum for twenty-four hours is collected 
in a suitable receptacle, and should be free from admixture of 
antiseptics. 

The sputum is examined in bulk with reference to its quan- 
tity, reaction, consistence, air-content, apparent composition, 
color, and odor. 

PHYSICAL AND CHEMICAL CHARACTERISTICS. 

The amount of sputum voided in twenty-four hours may 
be very great. On the other hand, even when every effort is 
made to expectorate, very little is produced. Some phthisical 
patients, in spite of violent coughing, raise very littlei, and that 
is of a very tenacious quality. 

Scanty, or absent, sputum may be evidence of tlie first stage 
of bronchitis, asthma, laryngitis, or pleurisy, while in children 

(19) 



20 THE SPUTUM. 

under 6 or 7 years of age it is usually absent, because it is 
swallowed. 

Abundant sputum is of importance in a general way be- 
cause it denotes, in acute infectious conditions particularly, that 
nature is prompt to relieve the body of an abundant secretion, 
which, if retained, might cause serious consequences by further 
reducing the already diminished respiratory capacity. When 
large amounts of sputum are voided at short intervals, alter- 
nated with periods of practical absence, one may infer a tuber- 
culous, gangrenous, or bronchiectatic activity, or rupture into 
the lung of an abscess of the lung, liver, kidney, or subphrenic 
space. 

Macroscopic Appearance. — The gross appearance of sputum, 
apart from its general consistency, is largely dependent upon 
the inclusion of adventitious material, and is clinically described 
under the following heads : — 

The consistence of sputum may bear a certain relation to 
the amount when, if abundant, the consistence is lessened, and 
vice versa. This relation is by no means constant. 

Ordinarily, sputum is "slimy /^ It may, however, be serous, 
purulent, or bloody. The peculiar slimy characteristic of spu- 
tum depends upon the amount of mucus contained, while its 
stickiness depends, in part, upon the mucin and, in part, upon 
the proteid content. This is especially marked in lobar pneu- 
monia. 

(a) Watery^ or serous, sputum, which is frequently blood- 
tinged, occurs in pulmonary edema, and in catarrhal influenza. 
Gastric disorders in neurotic old people may give rise to a thin, 
watery expectoration of considerable quantity, which is partly 
regurgitated and partly hawked up. 

(b) Viscid, or sticJcy, sputum, which adheres to the bottom 
of the container, even when completely inverted, is somewhat 
characteristic of lobar pneumonia, but may also be seen in 
phthisis, pertussis, and in broncho-pnemnonia. 

(c) Mucoid sputum, is a clear, diffluent sputum resembling 
egg-albumin, composed chiefly of mucus, is observed in the 
early stage of pneumonia, bronchitis, and phthisis; at the ter- 
mination of an asthmatic attack ; in pertussis, pharyngitis, laryn- 
gitis, measles, and influenza. 



PHYSICAL AND CHEMICAL CHARACTERISTICS. 21 

(d) Muco-purulent spntum is composed of mucoid sputum 
in which occurs a varying number of streaks and masses of 
opaque, yellow or greenish pus. It is noted toward the end 
of measles, in pertussis, in resolving pneumonia, during phthisis, 
and in subacute and chronic bronchitis. 

(e) Purulent sputum, which is composed purely of pus, is 
rather rare, and, when observed, indicates rupture of a liver, 
kidney or subphrenic abscess, or a purulent pleurisy into the 
respiratory tract. Opaque yellow sputum, consisting largely of 
pus, is found in bronchiectasis, phthisical cavities, broncho- 
pneumonia, and in the chronic or later stages of acute bron- 
chitis. 

(f) Nummular sputum. Eing or coin-shaped masses of spu- 
tum, which sink immediately in water, occur at times in bron- 
chiectasis, chronic bronchitis or phthisical cavity. 

(g) Frothy sputum may be observed in bronchitis, bron- 
cho-pneumonia, and emphysema. Its most important relation 
is in pulmonary edema, in which condition it is full of air and 
resembles frothy soap-water. 

Color. — (a) Rusty sputum, due to evenly distributed 
altered blood, is generally indicative of lobar pneumonia, but 
may also be observed in tuberculosis pulmonalis. 

(b) Prune- juice expectoration, is a rather fluid expectora- 
tion discolored by altered blood, and is seen in gangrene and in 
cancer of the lung. 

(c) Currant-jelly sputum is said to be characteristic of 
cancer of the lung. 

(d) Black sputum. Sputum which is very dark or black 
streaked or specked is found in persons who have inhaled coal 
dust or smoke for long periods of time. It is sometimes seen 
in gangrene of the lung. 

(e) Yelloiu, or green sputum may be caused by abscess of 
the liver which has ruptured into a bronchus (bile pigment), 
and is also seen in some cases of pneumonia (altered blood), and 
in pulmonary infections with chromogenic bacteria. 

(f) Shreds and casts may be observed in the sputum of 
chronic bronchitis, diphtheria, and rarely in penumonia. Casts, 
unless large and branching (Fig. 7), are more apt to be found 
during microscopic search. Suspicious particles should be 



22 



THE SPUTUM. 



floated in water against a black background^ and teased out 
with needles. 

(g) Blood-streaked sputum. Sputum streaked or discol- 
ored with blood may be due to violent vomiting or coughing, 
diseased tonsils or leakage from an aortic aneurism. Under 
these conditions it will appear as light red, but slightly altered 




Fig. 7.— Buoxchial Cast, from case of Fibrinous Bronchitis 
IN service of Dr. Judson Dalaxd (Original). 



blood. It may be present as a sequel of hemoptysis or abscess 
of the lung in broncho-pneumonia, empyema or in bronchitis. 

If the blood is dark or black it may be due to pulmonary 
infarction. Most commonly hemoptysis is observed in phthisis, 
when it recurs intermittently for daj^s or weeks. Finally, it 
should be remembered that malingerers may simulate disease of 
the respiratory tract by sucking blood from wounds of the gums, 
lips, tongue, or cheeks. 

(h) Ilemorrliagic sputum is observed in traumatic or 
tuberculous hemorrhage, in hemorrhagic infarctions, and in lobar 



PHYSICAL AND CHEMICAL CHARACTERISTICS. 23 

pneumonia. Also , in tumors in or near the respiratory tract, 
and finally in congestion of the pulmonary circulation. 

Certain derivatives from the blood-pigment produce in the 
sputum shades very similar to that of blood — the rusty sputum 
of pneumonia, for instance. The coloring matter here is partly 
changed blood and partly a yellowish-red derivative of blood- 
pigment, about which little is known. Peculiar lemon- 
colored and grass-green shades are also frequently observed in 
pneumonic sputum. These likewise are due to changed blood- 
pigment. Such sputa respond to Gmelin's test for bile-pigment 
(see page 346). A peculiar light-brown sputum is sometimes 
observed in heart disease, particularly mitral, in which amor- 
phous blood-pigment is found encapsulated in the alveolar epi- 
thelium (heart-failure cells). A type of green sputum has been 
observed in cases of lung tumor; the nature of the pigment in 
question is as yet unknown. 

(i) Extraneous dis colorations. Other noticeable discolora- 
tions of the sputum are observed from the admixture of inhaled 
dust-particles. The Maclc sputum of miners, and the blue spu- 
tum of workers in ultramarine, are examples of this class. 

Finally, it should be remembered that the sputum voided, 
following the ingestion of certain food-stuffs, may be discolored 
from contamination with these occurring in the mouth and 
pharynx. A greenish discoloration of the sputum is sometimes 
the result of the activity of certain chromogenic bacteria, espe- 
cially the Bacillus virescensA Yellow and bluish sputa of prob- 
able bacterial origin have occasionally been observed. 

The reaction of fresh sputum is generally alkaline; it may 
occasionally be acid, and usually becomes so after standing for 
some time through decomposition resulting from bacterial 
growth. 

Air Content. — Sputum is often more or less foamy or 
frothy, due to the presence of air. Other things being equal, 
the content of air in the sputum is greater the finer the bronchi 
from which the sputum is derived. The consistence of sputum 
often has a bearing on this. The amount of air contained can 
easily be determined by comparing its specific gravity to that of 



1 Frick : Virchow's Archiv, vol. cxvi, 1889. 



24 THE SPUTUM. 

water. Air-containing sputum will float; airless sputum will 
sink. 

The odor of fresh sputum is rather characteristic, but in- 
describable. On standing it may acquire a disagreeably nauseat- 
ing odor from decomposition, resulting from contained bacteria. 
Freshly voided sputum has a very decided odor in bronchiectasis, 
purulent bronchitis, tuberculosis, gangrene of the lung, and lung 
abscess. The disagreeable odor here arises from the activity of 
putrefactive bacteria. Stagnation of the secretion in cavities 
favors decomposition. In this way the foul odor of the expecto- 
ration in consumptives may be imparted to the breath. Finally, 
it must be remembered that fecal vomiting and diseased condi- 
tions of the mouth may be responsible for the odor of the breath 
and sputum, as will also the ingestion of certain volatile drugs, 
including alcohol. 

HEMOPTYSIS. 

True hemoptysis means the expectoration of an appreciable 
amount of pure, or nearly pure, blood, not merely sputum tinged 
with blood. The amount of blood may continue small, and may 
persist for many days or it may, as in the case of rupture of an 
aortic aneurism, be sufficiently large to cause death in a short 
time. 

Before making a diagnosis of hemoptysis it is necessary to 
exclude blood coming from the nose, pharynx, lar}Tix, buccal 
cavity, and tonsils. 

The Common Causes of Hemoptysis. — (a) Pulmonary dis- 
ease, usually tuberculous. It may also occur in the early stages 
of lobar pneumonia, abscess, bronchiectasis, gangrene, and can- 
cer of the lung. 

(b) Cardiac disease. Here it may be the result of venous 
obstruction occurring in the course of valvular disease. This 
is a not uncommon cause of slight but long-continued bleeding. 

(c) Vascular disease. The most important condition is 
rupture of an aneurism into the respiratory tract. Leakage from 
the same cause may cause slight but persistent hemoptysis. 

(d) Diseases of the Hood. Hemoptysis may occur during 
the course of hemophilia, purpura, leukemia, scurvy, and severe 



MACROSCOPIC EXAMINATION OF SPUTUM. 25 

anemia. It has been noted occasionally in the course of some 
of the exanthemata. 

(e) Miscellaneous causes. Vicarious menstruation and 
hysteria. 

MACROSCOPIC EXAMINATION OF SPUTUM. 

Examination should be made both upon a white and upon a 
black background. Many sputa appear to the naked eye to be 
homogeneous — pure mucus, pure pus, pure blood ; but sometimes 
not only may the sputum of one patient vary, but differences 
may be noted in each expectoration. 

''Dittrich's plugs" are yellowish-white masses, the size of 
a mustard-seed, and are easily seen over a black background. 
They come from the smaller bronchioles in putrid diseases, 
especially in putrid bronchitis and pulmonary gangrene. Micro- 
scopically they are composed of clumps of bacteria and fatty 
acid crystals. They have an intense and very disagreeable odor. 
Somewhat similar plugs may be encountered in the sputum in 
follicular tonsillitis. These plugs should not be confounded 
with those little masses described by, and known as, Cursch- 
mann's spirals (see page 27), or with the characteristic cheesy 
masses seen in the sputum of pulmonary tuberculosis. 

Formations consisting of fibrin are encountered in certain 
infections, and should be easily recognized by their white color, 
tenacious consistence, and sometimes by their shape (casts and 
molds) . 

Foreign bodies are sometimes aspirated into the respiratory 
tract, where they may remain for years without causing any 
symptoms until a fit of coughing dislodges them and they appear 
in the sputum. 

Concretions, known as broncholiths, or pneumoliths, de- 
pending on their origin, are sometimes, though very rarely, 
formed in the lungs during chronic inflammator}^ conditions. 
These are accidentally coughed up, when they may be found in 
the sputum. Occasionally these stones may have their origin 
in the crypts of the tonsils, or they may be calcified lymph-nodes 
that have ulcerated into the lung. 



26 THE SPUTUM. 

MICROSCOPIC EXAMINATION OF SPUTUM. 

A microscopic examination of the sputum reveals the pres- 
ence of cells, elastic fibers, casts, spirals, crystals, and micro- 
organisms. It is advisable to examine first the fresh unstained 
sputum, as the presence of fungi or cr\^stals in the sputum, and 
the nature of man}^ of the cellular elements can be determined 
only in this way. Afterward dried and stained cover-glass 
preparations may be made for more minute and detailed study. 

Preliminary Examination. — This is necessary in order to 
locate suspicious particles which may be scattered throughout 
the large mass of sputum. This is made either with the unaided 
e3'e or with a hand-lens. A thin layer of sputum is necessary 
to successful examination. For this purpose a moderate-sized 
Petri dish and cover is much better than the flat pieces of glass 
ordinarily employed, which are uncleanly and difficult to handle. 
A small amount of sputum is placed on the inside of the cover, 
and the other half of the dish pressed down into this, the rim 
very successfully preventing the escape of excess of material. 

The Unstained Specimen. — For the purpose of isolating any 
characteristic particles, the sputum should be spread out in a 
thin layer in the dish, and the material teased out with needles 
or tooth-picks. Having located a likely particle, it is transferred 
to a clean slide and flattened out by pressing a cover-glass down 
upon it. This should be examined first by the low, and later by 
the medium, power objective. 

Appearance. — Most sputum consists, microscopically, of 
a ground-work or mucous matrix of indefinite structure and 
appearance, in which are imbedded a variety of microscopic 
objects, principally cells : — 

1. Pus cells. The number of these indicates, in a general 
way, the purulent nature of the specimen. The character of the 
corpuscles varies greatly. Their size is from 7 to 10 micro- 
millimeters, they appear to be more or less granular, are some- 
times distinctly pigmented, containing one or more irregular 
nuclei. The granules are composed, some of proteid, some of 
fat, and some of extraneous debris. 

2. Epithelial cells found in the sputum differ from the pus 
cells, being usually larger in size, and by exhibiting one rather 



MICROSCOPIC EXAMINATION OF SPUTUM. 27 

large vesicular nucleus. Various types of epithelia are met in 
the sputum, and their recognition is of considerable value in 
locating the origin of the expectoration^ although, many times, 
the conditions to which they have been subjected after being shed 
have so altered their appearance that little information can be 
gained from their study: (a) Squamous epithelia are derived 
from the mouth, the pharynx, and from part of the larynx, (b) 
The cylindrical epithelia are derived from the nose or from the 
smaller bronchi, and are seen as pear-shaped or oval cells, some 
of which possess cilia, (c) Pulmonary, or alveolar epithelia 
are oval and measure from 20 to 30 micromillimeters in diameter. 

3. ''Heart-failure' cells. These are either oval or round, 
pigmented alveolar cells. When numerous their presence is said 
to be indicative of chronic passive congestion of the lungs, usu- 
ally depending on the failing compensation of cardiac valvular 
disease. Their presence is, therefore, usually associated with the 
common signs in the lungs, which are indicative of failing com- 
pensation, viz. : moist rales, mucous expectoration, and cyanosis. 

4. Eosinophiles may occasionally be found in large num- 
bers associated with Charcot-Leyden crystals in the expectora- 
tion of bronchial asthma. 

5. Bed hlood-cells. The appearance of red blood-cells in 
the sputum will depend largely upon the length of time that 
they have been shed. As they grow old they become pale, shad- 
owy, and fragmented. The finding of a few red blood-cells in 
the sputum is of no diagnostic import. They occur naturally 
in large numbers in hemoptysis, and are constant and more or 
less abundant in all inflammatory diseases of the lungs, par- 
ticularly phthisis. 

6. Casts. These may vary in size from those which repre- 
sent molds of the trachea and larger bronchi (see Fig. 7) to those 
coming from the smaller bronchioles, and which are from 14 to 
11/2 inches long. These smaller casts are the more common, and 
when present usually require the use of the low-power objective 
to demonstrate them. These casts usually occur in the three 
following diseases: the largest in diphtheria, medium size in 
fibrinous bronchitis, and the smallest in lobar pneumonia. 

7. Curs dim ann's spirals consist of worm-like spirals 1 to 
2 centimeters long, and about 1 millimeter wide. They are 



38 THE SPUTUM. 

more or less opaque, and are usually found surrounded by a 
thick, clear mass of mucus. They frequently show a central, 
undulating, thread-like core around which are twisted, in a 
spiral manner, the mucous threads. Entangled in these spirals 
are usually eosinophiles and Charcot-Leyden crystals. The 
spirals occur frequently in the sputum of bronchial asthma, more 
rarely in phthisis, bronchitis, and in lobar pneumonia. Their 
presence may be of service in differentiating bronchial from 
other forms of asthma. 

Crystals are never found in the freshly formed sputum, 
but are indicative of stagnation of the material within the body 
or of decomposition after being expectorated. The crystals usu- 
ally met in the sputum are : (a) Crystals of fat or of fatty acids 
appear as long, slender needles, either singly or grouped into fine 
rosettes or sheaves. They are readily soluble in potassium hy- 
drate or in ether. This solubility is easily determined by allow- 
ing a little of either fluid to flow under the edge of the cover- 
glass while observing the crystals in question. 

(b) Crystals of calcium phosphate may be encountered 
under conditions of retention and stagnation. 

(c) Charcot-Leyden crystals are occasionally encountered, 
particularly in the expectoration of bronchial asthma, and are 
here accompanied by eosinophiles. They appear as colorless, 
elongated double pyramids, varying considerably in size. They 
are often so small that high magnification is required to reveal 
them. 

(d) Cholesterin crystals are but rarely seen in the sputum. 
They occur as transparent, colorless, rhomboidal platelets, with 
notched or irregular angles and ends. 

(e) Hemato'idin crystals are derived from hemoglobin by 
a process of decomposition, and occur as needles and rhomboidal 
platelets of reddish and brownish hue. They are found chiefly 
in the sputum from old abscesses or perforating empyemas. 

(f) Leucin glohules and tryosin crystals are found in 
putrid sputum from old perforating abscesses or in putrid bron- 
chitis. 

(g) Calcium oxalate, in minute octahedral crystals, is 
occasionally met. 

Elastic Fibers. — When the lung-tissue is destroyed to 



STAIN FOR ELASTIC TISSUE IN SPUTUM. 29 

any extent by pathologic processes^ elastic fibers are likely to be 
encountered in the sputum. Their presence in the sputum proves 
conclusively the existence of some destructive process within 
the lung. Hence their importance in the diagnosis of tuber- 
culosis of the lung before the appearance of tubercule bacilli. As 
the healing process begins and progresses this elastic tissue grad- 
ually diminishes in amoimt, so that a constant presence or an 
increase in the amount indicates a progressive condition. Elas- 
tic tissue is also seen in abscesses of the lung, in bronchiectasis, 
in pulmonary infarct, occasionally in pneumonia, and in cases 
of gangrene of the lungs. 

These fibers may usually be detected in a thin layer of 
sputum examined microscopically with the medium or low 
power. Care must be observed to avoid confusing true elastic 
fibers with the somewhat similar vegetable fibers, which latter 
are generally larger and less uniformly wavy. 

Methods of Separating Elastic Tissue for Examination. — 
To detect particles or shreds of elastic tissue in the sputum, 
suspicious lumps should be thoroughly mixed with an equal 
quantity of 20 per cent, sodium hydrate solution; then a large 
volume of water added, and the whole allowed to sediment for 
a few hours. The sediment is then removed and examined under 
the microscope for the characteristic fibrillated masses. 

If the elastic tissue is not found by this procedure, the 
entire quantity of the twenty-four-hour specimen should be 
boiled with an equal quantity of 20 per cent, sodium hydrate 
solution. The resulting gelatinous mass is then mixed with 
several volumes of water, and allowed to sediment, the super- 
natant fluid poured off and about 15 cubic centimeters of the 
sediment centrifuged for fifteen minutes. The final precipitate 
is now carefully removed and examined as above. 

STAIN FOR ELASTIC TISSUE IN THE SPUTUM. 

Elastic tissue may be demonstrated by the orcein stain of 
Unna-Tanzar (see Appendix p. 482). In using this stain the 
suspected material is treated with a few cubic centimeters of the 
dye on a slide and then warmed for five minutes, after which the 
preparation is decolorized with acid alcohol. The elastic tissue 
fibers will be stained a brownish violet by this process. 



30 



THE SPUTUM. 



RARER DIAGNOSIS MADE BY EXAMINATION 
OF THE SPUTUM. 

Occasionally evidence of disease adjacent to the respiratory 
tract may be obtained by an examination of the sputum. Thus 
fragments of tumors occasionally appear in the specimen, which, 
if removed and prepared for section and staining, on examina- 
tion may clear up an obscure diagnosis. 




Fig. 8.— Actinomyces Showing Radial Formation. 

Pulmonary Actinomycosis. — This is a rather rare disease 
caused by the ray-fungus or actinomyces (see Fig. 8), The 
characteristic yellowish or grayish-green granules, if found, are 
often suflScient for a diagnosis; their composition should, how- 
ever, always be confirmed by microscopic search. In some cases 
the characteristic microscopic rosettes with clubbed rays are 
found; in others only branching threads, staining by Gram's 
method, will be found. These may be confused with atj^pical 
forms of the tubercle bacillus. 

EchinococcTis. — Earely echinococcus booklets enter the pul- 
monary tract and appear in the sputum. They usually originate 



PREPARATION OF THE STAINED SPECIMEN. 31 

in abscesses of adjacent organs, particularly the liver. (See 
section, ^Tarasites," page 188.) 

Distomnm Pulmonale; Syn. Distomum Westermanni. — This 
organism (for classification see page 179) is a not uncommon 
cause of disease of the lung in eastern countries, particularly in 
Asia, but may occasionally be encountered in other parts of the 
world. The ;Symptoms are not unlike those of pulmonary 
tuberculosis, for which it may be mistaken. Its presence is 
determined by finding the ova in the sputum. These are oval, 
of a brownish-yellow color, with a fairly thin shell, and meas- 
ure 0.0875 to 0.1025 millimeter in length, and 0.052 to 0.075 
millimeter in breadth. 

PREPARATION OF THE STAINED SPECIMEN. 

Any suspicious particles should be removed from the mass 
of sputum, and transferred to and carefully spread upon clean 
slides. These should then be treated to fixation and staining, 
the technic of which will depend upon the nature of the infor- 
mation sought. 

Microscopic Examination of Stained Specimens. — From the 
standpoint of clinical medicine the most important microscopic 
object for which search is made is the tubercle bacillus. 

Staining Peculiarities of the Tubercle Bacillus. — The recog- 
nition of the tubercle bacillus depends upon a special method 
by which they alone are stained. Unstained, tliey cannot usually 
be differentiated from the other organisms which may be pres- 
ent. The ordinary methods employed for staining bacteria are 
not suitable, so that special technic has been devised and is now 
regularly employed to render their recognition less difficult. 

In these methods advantage is taken of the fact that cer- 
tain substances increase the activity of staining by aniline dyes. 
With the tubercle bacillus this is accomplished with carbolic 
acid. Another important point is that these organisms, when 
once stained, give up their color only with great difficulty, so 
that agents which will decolorize all other bacteria in the course 
of a few minutes will have no appreciable effect upon the 
tubercle bacillus. It is upon these two peculiarities that we rely 
in differentiating this organism. 



32 THE SPUTUM. 

Differential Dia^osis. — While the peculiar micro-chemical 
reaction toward staining reagents is nsnally considered to be 
unique with the tubercle bacillus, it should be remembered that 
at least three other species of bacteria, when similarly treated, 
react in the same way. This fact is particularly important in 
connection with the microscopic examination of urine and patho- 
logic secretions from the genito-urinary tract, and from the rec- 
tum. Here is commonly encountered the smegma bacillus, which 
is the next most important member of the group of acid-fast 
organisms. Acid-fast bacilli have been found in the sputum 
and about the teeth and tonsils in a case of non-tuberculous dis- 
ease of the lung.2 

While these other organisms have the same acid-fast prop- 
erty as has the tubercle bacillus, they appear so seldom that seri- 
ous mistakes are not likely to occur. 

Special Staining Methods. — Ziehi^Nielsen : Place a few 
drops of the carbol-fuchsin solution (for preparation of stain see 
Appendix) upon the fixed cover-glass preparation, and hold 
over a low Bunsen flame until steam begins to rise. Do not boil. 
Continue the steaming process for from three to five minutes, 
pour ofl excess of stain, and wash in water. It is important to 
prevent the staining reagent from reaching the under surface 
of the cover-glass. If this is permitted the pigment will dry and 
become burned into the glass, when it will successfully resist all 
efforts at complete decolorization. Decolorize with acid-alcohol 
solution (see Appendix) or with 25 per cent, sulphuric acid. 
Decolorization should be continued for not less than ten min- 
utes or until the red color has entirely disappeared from the 
specimen; if this process is not thoroughly done the finished 
slide, so far as its diagnostic value is concerned, will be worth- 
less. The specimen is then washed in water, and counter-stained 
for from one to three minutes with a 1 per cent, aqueous solu- 
tion of methylene-blue, after which it is washed, dried, and 
mounted for microscopic examination. By this method the 
tubercle bacilli (see Plate I) will appear as characteristic red 
rods upon a blue background. A field which reveals other red 
objects has been insufficiently decolorized and should be dis- 
carded for a more carefully prepared specimen. 

2 Pappenheim : Berlin, klin. Wocli., No. 37, p. 809, 1898. 



PLATE I 




Tubercle Bacilli in Sputum. 



PREPARATION OF THE STAINED SPECIMEN. 33 

Gabbett's. — Flood the dried and fixed specimen with car- 
bol-fuchsin and steam for three minutes; then pass directly to 
the acid methylene-blue stain (see Appendix) for one minute. 
Finally wash, blot, dry in the air, and mount. This field, if 
properly prepared, should have the same appearance as that 
prepared by the Ziehl-Nielsen method, but is not as reliable as 
the preceding, because a dense spread may resist decolorization, 
which, owing to the deep-blue counter-staining, may not be 
noticed until the specimen is mounted for examination. 

Pappenheim recommends the following technic: 1. Stain 
in carbol-fuchsin by steaming near the boiling point for three 
or four minutes. 2. Pour off the excess of carbol-fuchsin and 
treat without washing with Pappenheim^s solution (see Appen- 
dix), pouring it slowly three or four times over the preparation, 
and allow it to drain off. 3. Wash in water, dry, and mount. 
Duration of entire procedure from three to five minutes. 

CzAPLEWSKT^s^.— This method employs the following solu- 
tion: (a) A special carbol-fuchsin (see Appendix). (&) 
Ebner's decolorizing solution (see Appendix). 

Technic. — 1. Stain, with the aid of heat, for three or 
four minutes. 2. Decolorize by treating the specimen alter- 
nately with Ebner's fluid and distilled water. 3. Counter-stain 
with methylene-blue for one minute. 4. Wash, dry, and mount 
in Canada balsam. The chief advantage of this method is said 
to be that all acid-resisting bacilli but the tubercle bacillus are 
decolorized, and, therefore, other organisms, such as the smegma, 
the hay bacillus, etc., will not, in a properly prepared specimen, 
appear as taking the special stain. 

Microscopic Appearance. — ^The tubercle bacillus is a delicate 
rod usually appearing in stained specimens with a beaded in- 
ternal structure. It may be straight, but is usually slightly 
curved in its long axis. The length is very variable, some being 
short, others quite long, though they never appear as long 
threads. The average length varies between 2 and 5 micro- 
millimeters, it is usually very slender. 

In sputum the organisms may occur singly or in groups of 
from three to half a dozen or more. 



8 Hyg. Rundschau, No. 21, 1896. 



34 THE SPUTUM. 

Clinical Significance. — The finding of tubercle bacilli 
in the spntnm is positive evidence of pulmonary, bronchial, or 
laryngeal tuberculosis. On the contrary, their absence, after 
careful search, even, of a number of preparations, cannot be 
considered absolute negative evidence. 

SPECIAL METHODS OF CONCENTRATING SPUTUM TO 
FACILITATE BACTERIAL EXAMINATIONS. 

If, after careful search of suspicious particles taken from 
the sputum of a suspect, whose clinical history and symptoms are 
strongly suggestive of the disease, the organism is not found, 
the following devices may be resorted to: — 

A. The whole amount of a twenty-four hours' specimen of 
sputum, to which no antiseptic solution has been added, should 
be placed in a porcelain dish or glass beaker, and stirred with a 
glass rod until quite thin and diffluent. This should then be 
stood aside for a few hours to settle, when the lowest portion 
of the fluid is taken up in pipette and transferred to a centri- 
fuge-tube, where it is rotated for from fifteen minutes to half 
an hour. From the bottom of the centrifuge-tube a small 
amount of the sediment is transferred to a cover-glass or slide 
for fixing and staining. 

B. A considerable quantity of sputum is mixed with an 
equal quantity of water, and a few drops of a 10 per cent, solu- 
tion of sodium hydrate added. This mixture is then heated 
until homogeneous, when it is cooled, sedimented, and centri- 
fuged, and examined as outlined above. 

ISOLATION OF GRANULAR FORMS OF TUBERCLE BACILLI. 

Some observers have found that some specimens after a 
most careful concentration may fail to show the characteristic 
organisms by the differential stains, yet they may still, by a 
more or less elaborate technic, be isolated. These are the so- 
called granular forms of tubercle bacilli, which are considered 
degenerative products of the common forms of the organism. 

Seelas,^ however, states that he has never missed finding 
bacilli themselves in cases where the ^^granules^' can be detected. 



4 Semaine medicale, April 30, 1913. 



ANTIFORMIN METHOD. 35 

In examining for the granules he adds to the sputum 2 
drops of 10 per cent, sodium hydrate solution, shakes a glass- 
stoppered bottle, heats to boiling, adds one-third part of 50 per 
cent, alcohol, and allows the mixture to stand in a conical glass 
for a few hours, after which the upper two-thirds are decanted 
and the remainder subjected to centrifugation at 4000 revolu- 
tions a minute. The flocculent deposit is then placed on slides 
previously cleansed with potassium permanganate, concentrated 
sulphuric acid, and absolute alcohol. Two slides are slid one 
over the other until the deposit has become dry. Fixation is 
then secured and the specimen stained by the Ziehl-Nielsen 
method. The staining is done with the slides on a warm copper 
plate. Finally, the preparations are washed in running water, 
and counter-stained with Loeffler's methylene-blue ; both the 
tubercle bacilli and "the granules" will appear as red bodies on 
a blue field. 

Antiformin Method. — The most notable advance in the 
microscopic examination of sputum for tubercle bacilli is the 
development of the antiformin process of Uhlenhuth.^ 

Antiformin is a 10 per cent, solution of sodium hypochlorite 
containing from 5 to 10 per cent, of sodium hydrate. This 
preparation may now be obtained as a proprietary in the open 
market. 

The technic originally advised by Uhlenhuth has under- 
gone slight modifications since it was first published. The 
technic of E. Burvil Holmes (personal communication) is as 
follows : — 

"If the specimen of sputum is not large I mix it with an 
equal quantity of a 20 per cent, solution of antiformin in any 
sterile receptacle, usually a large test-tube or, better, centrifuge 
tube. This is thoroughly shaken until all the nummular masses 
are dissolved. The mixture will take on a brownish color when 
this is complete. A little alcohol (95 per cent.) is now added, 
as I have found that without it, owing to, I presume, the different 
densities of the two substances, the bacteria are not well thrown 
down. The tube is then placed in an electric centrifuge and 
centrifuged for ten minutes at high speed. The mixture, except- 
ing that at the very bottom, is pipetted off, and some normal 

s Berlin, klin. Med., August 29, vol. v, 35. , 



36 THE SPUTUM. 

NaCI is added to the small portion at the bottom of the tube 
and this is thoroughly mixed and again centrifnged for five 
minutes. Again all except the very bottom, which is placed on 
a clean glass slide upon which preferably a little egg-albumin 
has been smeared, is pipetted off. The slide is stained in the 
usual way.^^ 

Another modification is that advocated by Webster,^ which 
is Loeffler^s" modification of the original antif ormin process. The 
technic is as follows : 5, 10, or more cubic centimeters of sputum 
are placed in a flask and mixed with an equal quantity of a 50 per 
cent, solution of antif ormin (a 10 per cent, solution of sodium 
hypochlorite containing 5 to 10 per cent, of sodium hydrate) 
and boiled for a period not exceeding fifteen minutes. Solu- 
tion occurs associated with considerable foaming and browning 
of the mixture. For every 10 cubic centimeters of this solu- 
tion are now added 1.5 cubic centimeters of a mixture of 1 part 
of chloroform and 9 parts of alcohol. After thoroughly shak- 
ing to produce a fine emulsion, portions of the fluid are placed 
in sedimenting tubes, the tubes are corked, and centrifuged for 
fifteen minutes. The heavier elements collected in a film just 
above the chloroform, which film holds the tubercle bacilli, 
owing to the marked affinity of chloroform for the fatty and 
waxy material in these organisms. The supernatant liquid is 
poured off and the film above mentioned is removed and placed 
upon a glass slide, the excess of fluid being taken up with filter- 
paper. As a fixative a drop of egg-albumin, preserved with % 
per cent, carbolic acid, is added and a thin spread made by 
means of a second slide. This smear is allowed to dry and is 
then stained by one of the usual methods. 

This enrichment process of Loeffler furnishes preparations 
which often show a remarkable increase in numbers of tubercle 
bacilli as compared with those found by the usual smear 
methods. A further advantage of this method is that prac- 
tically all organisms, with the exception of those of the acid- 
fast type to which the tubercle bacillus belongs, are destroyed. 
This permits one. to obtain material (omitting, of course, the 
application of heat) for pure-culture work or for inoculation 

« "Diagnostic Methods," P. Blakiston's Sons, Philadelphia, 1912. 
7 Deutsch. med. Wochen., Bd. 36, 1910, S. 1987. 



ELLERMANN-ERLANDSEN'S METHOD OF STAINING. 37 

purposes, which will be free from the secondary invaders which 
so often interfere with the establishment of an absolute diag- 
nosis. ISTaturally, in the study of the mixed infections in 
tuberculosis one should also examine preparations made in the 
usual way. 

Method of Ellermann-Erlandsen.s — Another method for 
concentrating tubercle bacilli in a specimen is H. KogePs modi- 
fication of the so-called double method of Ellermann-Erlandsen 
and is as follows: (1) One volume of sputum (10-15 cubic 
centimeters) is mixed in a stoppered glass bottle with one-half 
its volume of 0.6 per cent, sodium carbonate solution. The 
mixture now stands twenty-four hours in the thermostat at 
37° C. (2) The greater part of the supernatant fluid is 
decanted and remainder is centrifugated in a graduated centri- 
fuge tube. The fluid is poured off. (3) Four volumes of 0.25 
per cent, sodium hydroxide are added to one volume of the 
precipitate. After very carefully agitating, this is raised to 
boiling. (4) Centrifuge and make smear of the sediment. 

The result of this treatment is that practically the entire 
bacillary content of the whole amount of sputum is spread on 
one or two slides and 20 to 30 times the number of tubercle 
bacilli occur per field. The method takes time. In an old 
sputum autodigestion may have gone far enough to make the 
first stage of the procedure unnecessary. Very thick sputa may 
require 48 hours in the thermostat. Very purulent sputa give 
poorer results than slimy ones. These must be left longer in 
the incubator and must be boiled longer, with larger amounts of 
the caustic. A powerful centrifuge is required, and the centrif- 
ugation must be continued long enough to precipitate com- 
pletely all the solid matter. The final precipitate consists almost 
entirely of bacteria. A glance is often all that is required for a 
diagnosis. Specimens which in the usual smear showed 10 
tubercle bacilli to the field, by the double method showed 300 
to 400. As a rule, 15 to 30 times as many may be seen. Of 105 
specimens, of sputum examined by the usual method, 21 were 
positive. Of those negative, 8 by the double method gave posi- 
tive results, or an increase of 8 per cent. 



8 Deutsch. med. Wochen., Dec. 2, 1909. 



38 THE SPUTUM. 

Much's Method of Staining. — Among the newer methods of 
staining tubercle bacilli is the method of Much, which has been 
carefully studied by Liebermeister,^ who says that certain tubercle 
organisms not demonstrated by the usual methods may be 
shown by a prolonged Glram stain; the tubercle bacilli being 
G-ram-positive though they are not absolutely acid-fast. The acid- 
fast types of tubercle bacilli are distinctly granular and frequently 
appear as mere "granules" rather than true bacilli (see page 34). 
These "granules" under certain unknown conditions change 
into true bacillary types and vice versa. Smears are prepared 
in the usual way or by one of the special methods above de- 
scribed, and are then treated as follows : Cover the smear with 
carbol-methyl-violet solution (Much's stain, see Appendix 
page 500), and heat to boiling several times. Wash stain off 
with water and cover smear with Lugol's solution for five min- 
utes. Wash with water and treat with 5 per cent, nitric acid 
for one minute and follow this with 3 per cent, hydrochloric 
acid for ten seconds. Without washing place the slide in a 
mixture of equal parts of acetone and absolute alcohol until 
the smear is colorless. Wash with distilled water and counter- 
stain with 1 per cent, aqueous solution of saffranin for a few 
seconds. Wash in water, dry thoroughly and examine with im- 
mersion lens. The tubercle bacilli and the granular forms 
appear blue, while the other organisms are red. 

Spengler's technic is fairly simple and is applied to the 
dried film as usually prepared: — 

1. Stain with carbol-fuchsin, warm, but without too much 
heat. 2. Pour off the stain without washing. 3. Pour on picric 
acid alcohol (consisting of equal parts of saturated aqueous 
solution of picric acid and absolute alcohol). After three 
seconds: 4. Wash with 60 per cent, alcohol. 5. Treat with 
15 per cent, nitric acid till yellow (thirty seconds). 6. Wash 
again with 60 per cent, alcohol. 7. Counter-stain with picric 
acid alcohol till lemon-colored. 8. Wash with distilled water 
and dry gently at a low heat. 

The bacilli appear bright red on a lemon-colored ground, 
and, if present, are more perceptible than by any other method. 
With a little practice this will be found an easy and fairly 

» Deutsch. med. Wochen., July 15, 1909. 



MICROCOCCUS LANCEOLATUS. 39 

rapid method, and the extra time involved in the process will 
be more than compensated by the ease with which the bacilli will 
be found, if present. ^^ 



MICROCOCCUS LANCEOLATUS. 

The presence of FrankePs diplococcns in the sputum of 
patients suffering with croupous pneumonia is fairly constant, 
so that its demonstration is of considerable diagnostic impor- 
tance. They appear as elongated lanceolate cocci, usually 
arranged in pairs with their bases approximated. They are sur- 
rounded with a faintly staining capsule which, in dry prepara- 
tions, does not usually take the stain at all, although the ordi- 
nary method employing methylene-blue has occasionally, in the 
author's experience, demonstrated a faint but distinct capsule. 

The organism is supposed to be the cause of lobar pneu- 
monia, but must not be confounded with the other diplococci 
occurring in the sputum, more especially with Friedlander's 
bacillus. The latter also possesses a capsule, but has nothing 
whatever to do with the production of lobar pneumonia, though 
occasionally they may accidentally be present. Friedlander's 
organism, when highly magnified, will be found to be a short 
rod. Cultural characteristics will also serve to differentiate, 
as will also Gram's staining method, which decolorizes Fried- 
lander^s and stains Fr ankers organism. 

The following modification of Gram's method will be 
found satisfactory (for preparation of staining reagents see 
Appendix) : Hold the fixed and dried cover-glass preparation 
in the forceps and flood with carbol-gentian violet; allow this 
stain to act for from three to four minutes. Wash and trans- 
fer to the iodine-potassium-iodide solution for from one to two 
minutes. Next wash in alcohol until the apparently dirtily 
stained film is decolorized. Transfer to absolute alcohol, then to 
oil of cloves, and finally mount in balsam. By this method the 
organism appears as a dark-blue or violet diplococcus. Fried- 
lander's organism will remain unstained. 

A ver}^ useful method for differentiating Friinkel's coccus 
is that devised by W. Wolf. By this method the dry preparation 
is first stained in aniline water saturated with fuchsin, and is 
then placed for one or two minutes in a dilute watery solution 



40 THE SPUTUM. 

of methylene-blue. The cocci will now be found stained blue, 
the capsule rose-red. and the body of the specimen purplish red. 

Method for Staining Capsule. — Prepare the cover- 
glass smear in the usual way, then without drying flood with 
glacial acetic acid. At the expiration of one minute pour off 
the excess of acid, and without washing flood the specimen with 
aniline water gentian-violet (Koch-Ehrlich)^ which should be 
allowed to act for four minutes, when the excess of stain is 
poured off and a fresh portion added, which is allowed to act 
for another two minutes. The cover-glass is now washed in two 
or three changes of normal saline solution (it may be found 
necessary to employ a saline solution of 1.5 to 2.0 per cent.), 
after which it is blotted, dried, and mounted in the usual way. 

By this method, the capsule will appear as a faintly tinted 
halo about the diplococcus. Clinically the absence of FrankePs 
micro-organism practically excludes the diagnosis of lobar pneu- 
monia, although its demonstrated presence is by no means posi- 
tive evidence in the other direction^ because this organism has 
repeatedly been demonstrated in the sputum and in the mouth 
secretions of healthy individuals. 

i/^ BACILLUS OF INFLUENZA, OR PFEIFFER'S BACILLUS. 

A small, slender bacillus occurring usually in very great 
numbers in the nasal secretion of fresh attacks of true influenza, 
but not found in the ordinary short attacks of prostration accom- 
panied by coryza, which is at present designated influenza or 
^%rippe,^^ largely for lack of a more definite diagnosis. Mor- 
phologically, it is a very small rod appearing frequently in pairs ; 
in parts of the secretion this organism may be found in what is 
practically a pure culture, occurring both within and without 
the leukocytes. Their length is usually from two to three times 
their width; they rarely form chains. The ends of the rods are 
rounded, and if imperfectly stained (they stain with diffi- 
culty) the ends will be more deeply stained than the center, 
giving an appearance not unlike a diplococcus with just a sug- 
gestion of a capsule. According to recent authority, this bacillus 
does not possess a capsule, the deceptive appearance being prob- 
ably a staining peculiarity of certain cells. The bacillus is non- 



LEPRA BACILLUS. 41 

motile, and can only be cultivated "upon special media con- 
taining hemoglobin. This characteristic will readily serve to 
differentiate it from the colon bacillus and other organisms of 
similar appearance. An easily made and satisfactory medium is 
prepared by spreading a little fresh blood upon the surface of 
an ordinary agar slant and inoculating this with the infected 
material. This organism only develops on artificial media be- 
tween the temperatures of 26° and 43° C., growing best at body- 
temperature. Upon the blood-agar slant incubated at 37° C.^, 
there will develop minute, transparent, watery colonies that are 
without structure, somewhat resembling droplets of dew. They 
are usually discrete, and show little or no tendency to coalesce. 

Staining. — One of the best methods of staining is with a 
dilute watery solution of ZiehPs carbol-fuchsin (the color of 
the solution should be pale red). This solution should be al- 
lowed to act for five minutes. This organism is decolorized by 
Gram's method. 

A second method of staining is with Loeffler's methylene- 
blue (for preparation see Appendix). This stain should be 
allowed to act for five minutes. Then wash in water, mount, 
and examine for blue organisms. 

LEPRA BACILLUS. 

The bacillus lepras, first described by Hansen, is a small, 
slender bacillus from 4 to 6 microns in length and surrounded 
by a slimy envelope. These bacilli behave toward staining re- 
agents very much like the tubercle bacillus, but are less resistant 
toward acid and alcohol than is the tubercle bacillus, so that 
a differentiation is possible, provided decolorization is rather 
severe. The stained bacillus often shows clear spots or appears 
as if made up of stained granules. 

Methods of Staining. — Any of the special stains employed 
in examining for the tubercle bacillus may be used. Of these 
that of Pappenheim is very satisfactory, because, when decolor- 
ization is complete, nothing retains the red pigment but the 
tubercle bacillus. Such staining methods applied to the lepra 
bacillus are at best of negative value. 

These organisms may be found in many cases of leprosy, in 
the sputum or in the nasal secretions, so that in doubtful cases 



42 THE SPUTUM. 

a differentiation is necessary. It may be necessary to resort to 
inoculation experiments to make an absolute differentiation. 

THE BACILLUS PERTUSSIS. 

This organism, discovered by Bordet and Gengou, and 
elaborated by Klimenko, has been frequently found in the 
sputum in cases of whooping-cough. It resembles very closely 
the influenza bacillus^ appearing as short, plump, ovoid bacilli, 
with rounded ends, lying singly or in small groups between the 
pus and epithelial cells. It stains feebly with the usual dyes and 
is Gram-negative. This organism is rarely intracellular and 
may thus be distinguished from the influenza bacillus. 

SPECIAL REACTIONS APPLIED TO THE SPUTUM. 

In recent years several chemical tests and reactions have 
been applied to the sputum, as further aids in diagnosis, some 
of which have already attained a practical value. 

Pacini's Color Reaction in Pneumonia. — A. P. J. Pacinii® 
describes a reaction persistently observed in the sputum of 
pneumonia patients which, aft-er examination of 1200 speci- 
mens where the ultimate diagnosis was confirmed and estab- 
lished as pneumonia, showed an error of only 2 per cent. 

Because of the combined accuracy and simplicity of the 
reaction, it should prove useful to the busy general practitioner. 

This test is applied as follows : A portion of the sputum is 
mixed with distilled water in the proportion of one volume of 
sputum to ten volumes of water, and agitated in a suitable 
container for five minutes. The mixture is filtered through 
paper and preserved for the test. 

A 1 per cent, aqueous solution of methyl-violet constitutes 
the reagent necessary for this reaction, which should be prepared 
as stock and kept ready for use. 

To a test-tube containing 10 cubic centimeters of distilled 
water add 5 drops of methjd-violet solution and mix thoroughly. 
Then add, drop by drop, 10 drops of the filtrate obtained as 
above described. 

In the event of a positive reaction, the methyl-violet 



10 The Interstate Medical Journal, June, 1913, p. 536. 



SPECIAL REACTIONS APPLIED TO SPUTUM. 43 

assumes a distinct red color. Nothing short of a red color con- 
stitutes a reaction. 

This reaction is present only in the sputum of patients at 
the onset of or during an attack of pneumonia. It is due to a 
specific disintegrated blood pigment characteristically present in 
the sputum of such patients, and precedes the expectoration of 
the classic "rusty sputum^^ by several days. 

Test for the Albumin Content of the Sputum. — Beginning 
with the work of Eodger and Levy-Yalanci,ii and since re- 
peated by others, it has been found that the sputum of tuber- 
culosis in nearly every case, irrespective of age, gives a positive 
albumin reaction, which substance is not normally present in 
the sputum. It is now believed that the demonstration of albu- 
min in the sputum is a most important diagnostic aid in the 
demonstration of the earlier phases of pulmonary tuberculosis, 
even before the appearance of the specific bacilli in the sputum. 
No direct relation between the number of tubercle bacilli present 
and the albumin contained has yet been demonstrated. Some 
cases give negative reaction and fail to show the germs, after 
most careful examination, and vice versa. 

According to Dr. John Eitter,i2 a good technic is as follows : 

Test for Sputum Albumin. — A proper way for collecting 
sputa is most essential to the correct interpretation of the albu- 
min reaction. First wash out the mouth or gargle with a few 
mouthfuls of warm water to clear the mouth and throat from all 
adherent secretions, after which the sputum should be col- 
lected. The patient must be instructed to cough up deep from 
the lungs, usually in the early morning, the first on rising, and 
place the sputum in a clean sterile bottle and securely close it 
with a well-fitting cork stopper. He must be specifically in- 
structed not to collect the secretions from the mouth, buccal 
cavity, nasopharynx, or from the throat, but only such as he can 
bring up from the bronchial tubes, for this alone is suitable for 
making a reliable albumin test. A properly collected specimen 
should be examined at once; in any event it must not be more 
than about six hours old ; in cold weather an expectorated sample 
will keep for twenty-four hours before undergoing fermentative 

iiPresse medicale, No. 32, 1910; No. 40, 1911. 
12 Medical Record, April 26, 1913. 



44 THE SPUTUM. 

changes. Only the freshly collected sputum should be tested; 
decomposed secretions will often give a reaction simulating albu- 
min. No antiseptic solution or any preservative agent should be 
added before making the albumin test. 

TeChnic. — Put 5 cubic centimeters of sputum into a glass 
cylinder of about 25 cubic centimeters' capacity (glass cylinder 
must be supplied with a well-fitting ground-glass stopper), add 
5 cubic centimeters of distilled water and about 10 drops to 1 
cubic centimeter of glacial acetic acid, replace the stopper, shake 
well, and set aside. Shake occasionally during the next twenty 
or thirty minutes, then proceed to filter. If the sputum is suit- 
able for filtration it will have separated into three distinct 
layers, reminding one very much of an expressed stomach con- 
tent ready for testing. Should the middle or watery portion of 
the sputum solution be still a little opaque, showing the presence 
of some remaining unprecipitated mucin, add a few drops more 
of the glacial acetic acid, again shake well, and set aside as be- 
fore. The fluid portion now appearing clear, proceed to filter 
through a plain wet filter and test the clear filtrate for albumin 
in the usual way by either Heller's test or the heat test. 

Rivalta's Acetic Acid Test for Albumin in Sputum as Modi- 
fied by Casali.13 — Because of its simplicity this method has much 
to recommend itself to the busy practitioner. The technic of 
this test is as follows: Ten cubic centimeters of sputum are 
thoroughly stirred with 10 cubic centimeters of distilled water 
and then slowly filtered. Two drops of acetic acid are stirred 
into a glass containing 100 cubic centimeters of distilled water. 
Holding the glass before a light, a drop of the filtrate is cau- 
tiously dropped into the fluid. In case of a positive reaction, 
the drop becomes surrounded with a little cloud as it falls 
through the fluid. By graduated dilution of the filtrate with a 
weak solution of sodium carbonate (1 drop in 100 cubic centi- 
meters of water), the test can be carried to its extremest limits. 
The significance of albumin found by this test is the same as in 
the preceding. 

13 Riforma Medica, Naples, July 27, 1911, No. 30. 



III. 

CLINICAL HEMATOLOGY. 



GENERAL CONSIDERATIONS. 

The blood is the most important fluid of the body, as it 
bears a more or less definite and direct relation to all other body 
fluids and tissues. It is the intermediate substance between the 
nutritive materials taken into the body through the digestive 
tract and elaborated into pabulum by it, and body cells, to which 
it both gives nutriment and carries away effete and waste 
materials. 

We must, therefore, have a working knowledge of the 
normal physiology and histology of the blood, in order to com- 
prehend departures from the normal and to appreciate their 
pathologic significance. 

There are but few definite diagnostic findings which can be 
based upon hematologic examination alone; nevertheless, there 
are an infinite number of conditions in which a study of the 
blood, from one or more standpoints, will shed valuable light, 
which will often give material aid in an obscure diagnosis. It 
must be remembered that both physiologic and pathologic con- 
ditions may influence both the quantity and the quality of the 
blood. This is becoming more and more evident as the mysteries 
of the plasma are being brought to light through recent sys- 
tematic studies of the agglutinins, opsonins, lysins, precipitins, 
the phenomenon of anaphylaxis, etc. 

It is beyond the scope and purpose of this book to cover 
completely the whole subject of clinical hematology, and no 
effort has been made to so do ; any omissions encountered by the 
student will have to be augmented by collateral reading from 
one of the recent works on the blood. The author's intention 
has been, in the preparation of this section, to furnish the active 
practitioner and student with a safe practical guide to the per- 
formance of the more commonly used clinical methods. 

(45) 



46 CLINICAL HEMATOLOGY. 

Technic requiring considerable time for its performance, 
or complicated apparatus, has largely been eliminated, while, 
as a matter of convenience, sections on the coagulation time and 
viscosity have been included under the general head of the blood. 

PHYSICAL AND CHEMICAL PROPERTIES. 

Appearance of Fresh Blood. — The exuding drop of blood 
shows even to the naked eye a number of properties. The redder 
it is, the richer it is in oxyhemoglobin; the darker, the greater 
the amount of reduced hemoglobin. Microscopically, it reveals 
a great number of cellular elements; these are colored and col- 
orless discs. 

The red cells appear as non-nucleated biconcave discs, 
measuring on the average 7 micromillimeters in diameter. 
Viewed singly through the microscope by transmitted light, they 
are of pale-greenish hue. The colorless cells or white corpus- 
cles are, as a rule, somewhat larger than the red cells, and pre- 
sent either mono- or poly- nucleated protoplasm. 

TTie plaques, or blood-platelets, appear as minute, colorless 
discs measuring less than half the diameter of the red cells. 
They usually occur in groups or bunches of half a dozen or 
more, and are present in normal blood to the numiber of from 
225,000 to 350,000 per cubic millimeter. 

There are no other morphologic constituents of the blood. 

Color. — The color of normal blood is due to the presence 
of an albuminous substance in the corpuscles termed hemo- 
globin. In the arterial blood it is in combination with oxygen, 
and is here termed oxyhemoglobin. In the venous blood a mix- 
ture of both hemoglobin and oxyhemoglobin occurs. With a 
preponderance of oxyhemoglobin, the blood tends to a scarlet 
hue; when the hemoglobin predominates, the blood is of a 
bluish color. 

Pathologic Changes in Color. — In coal-gas poisoning the blood is 
cherry-red. After poisoning from potassium chlorate, aniline, hydro- 
cyanic acid, and nitrobenzol, the blood is brownish-red or chocolate color. 
In extreme cases of leukemia the blood may have a milky appearance 
due to the excessive number of white blood-cells present. 



PHYSICAL AND CHEMICAL PROPERTIES. 



47 



The Odor. — This is characteristic and differs in different 
species of animals. It is due chiefly to the presence of volatile 
fatty acids. 

The Taste. — The taste of blood is salty, but at the same 
time insipid. 

The Specific Gravity. — ^The specific gravity seems to vary 
with the amount of hemoglobin. It is influenced by the age and 
sex of the individual, the process of digestion, exercise, preg- 
nancy, etc. The normal average in adults varies between 1.058 
and 1.062. 




Fig. 9.— Pycnometer. 



Method by Pycnometer. — The most accurate instrument 
for determining the specific gravity is the pycnometer. This 
method is open to the objection that it requires much more 
blood (10 to 50 cubic centimeters) than can usually be obtained 
in routine work. In cases in which bleeding can be resorted 
to without detriment to the patient, this is the method to use. 

The technic is as follows: Weigh the pycnometer (Fig. 9) 
three times, on an accurate clinical balance: (a) empty; (&) 
filled with distilled water, and (c) filled with blood. Care should 
be taken to have the vessel absolutely dry and clean before weigh- 
ing it empty and before filling with either water or blood. Sub- 
tract the weight of the empty bottle from that of the bottle 
filled with blood and divide this figure by the difference in weight 
between the bottle filled with water and the empty bottle. The 



48 CLINICAL HEMATOLOGY. 

result will be the specific gravity of the blood, water being taken 
as unity. In this determination it is essential, in order to in- 
sure accurate results, to have the temperature of the water the 
same as that of the blood. 

Method of Hammerschlag. — A cylinder about 10 centime- 
ters in height is partly filled with a mixture of benzol (sp. gr. 
0.889) and chloroform (sp. gr. 1.526), so that the specific 
gravity of the mixture lies between 1.050 and 1.060. Into this 
a drop of blood is allowed to fall directly from the finger. It is 
then brought into equilibrium by the addition of either a little 
chloroform or benzol, according to the tendency of the drop to 
sink or rise in the cylinder. 

As soon as the drop remains stationary in the fluid the 
specific gravity of this is taken by an accurate hydrometer (one 
reading to the fourth decimal should be used) or, better, with 
a Westphal balance. The reading represents the specific grav- 
ity of the blood tested. 

The Amount. — ^The total amount of blood in the normal 
adult is said to amount to about one-twelfth or one-fourteenth 
of the body weight. 

The Eeaction. — The reaction of the blood is slightly alka- 
line, due to the presence of the monosodium carbonate and the 
disodium phosphate in solution in it. The reaction may be 
roughly determined by drawing a strip of neutral litmus paper, 
which has been thoroughly moistened with a concentrated solu- 
tion of common salt, through the blood, and then rapidly wash- 
ing the corpuscles off with the same solution. Owing' to the 
development of certain acids, the alkalinity of the blood rapidly 
diminishes after it is shed. This fact renders this determina- 
tion a rather difficult matter. The normal variation of alkalinity 
is very slight. By accurate titration the normal degree of alka- 
linity of the blood, under normal conditions, corresponds to 
from 220 to 300 milligrams of sodium hydrate for every 100 
cubic centimeters of blood. 

Method of Engbl. — This is a practical clinical modifica- 
tion of the method of Lowy. The apparatus is self-contained 
in a case which can be conveniently transported. The blood 
is drawn directly into a special pipette which measures 0.05 
cubic centimeter and then allows a dilution with 5.0 cubic 



CHEMICAL COMPOSITION OF THE BLOOD. 49 

centimeters of distilled water. The mixture of blood and 
water is placed in a small glass beaker, and titrated with a N/75 
solution of tartaric acid. The end-reaction occurs when a dis- 
tinct red color appears at the edge of a drop of the test solution 
allowed to drop upon a piece of lacmoid paper. Calculation: 
1 cubic centimeter of 'R/25 tartaric acid is equal to 0.00053 
grams sodium hydrate. 

MiETHOD or LowY. — Measure accurately 5 cubic centim- 
eters of blood drawn from a vein and inject directly from the 
syringe into 45 cubic centimeters of 0.25 per cent, solution of 
C. P. sodium oxalate ; 5 cubic centimeters of this mixture is then 
placed in a small porcelain dish and titrated with a N/25 solu- 
tion of tartaric acid, using lacmoid paper, previously soaked in 
a concentrated solution of magnesium sulphate as an indicator. 

^"72 5 tartaric acid is prepared by dissolving 3 grams of 
C. P. tartaric acid in 1000 cubic centimeters distilled water. 

The alkaline equivalent of this solution is : 1 cubic centim- 
eter equals 0.0016 gram sodium hydrate. The number of cubic 
centimeters of the N/25 tartaric acid used multiplied by 0.0016 
will give the number of milligrams of alkali in I/2 cubic centim- 
eter of the original 5 cubic centimeters of blood used. 

THE CHEMICAL COMPOSITION OF THE BLOOD. 

A general idea of the composition of the blood may be 
had from the following table, which is taken from Simon's 
"Physiologic Chemistry.^' The calculations are made for 1000 
parts by weight. 

Corpuscles 480.00 parts 

Water 276.90 " 

Oxyhemoglobin 193.90 " 

Stroma, including salts 9.20 " 

Plasma 520.00 parts 

Water 477.37 " 

Albumins 35.88 " 

Extractives 2.39 " 

Inorganic salts 4.36 " 

The predominating solid substance in the blood is oxy- 
hemoglobin; it represents 10' per cent, of the total weight of 

4 



50 CLINICAL HEMATOLOGY. 

the blood, 40 per cent, of the weight of the corpuscles, and 65 
per cent, of all organic matter present. 

The mineral constituents comprise sodium, potassium, cal- 
cium, magnesium, and iron. 

Fats are present to the extent of from 0.2 to 0.3 per cent. 
These may be temporarily increased after the ingestion of much 
fatty food, and also in many pathologic conditions. 

The plasma normally contains small amounts of oxygen 
and nitrogen in solution, with varying amounts of carbon 
dioxide. 

METHODS OF OBTAINING BLOOD FOR EXAMINATION. 

The tip of the finger and lobe of the ear are the sites usually 
selected from which to obtain specimens. In the majority of 
examinations only a small amount — a few drops — is necessary. 
This is obtained by simple puncture of the skin made with a 
Glover needle, the half-point of a new steel pen, a Daland 
lancet (see Fig. 10), or the so-called pistol-knife. The last- 
mentioned two instruments are to be preferred because they 
permit of regulation of the depth of the puncture. 

For hospital and laboratory use, in order to assure a sterile 
instrument for each patient, I have, for a number of years, 
employed short sections of thin glass tubing, drawn out in the 
flame to a capillary point, and then broken so as to have a 
short, sharp end. There is little danger, if these are properly 
made, of leaving fragments of glass in the tissues. After 
using, these may either be thrown away or retipped in the 
flame. 

For larger quantities of blood, a few cubic centimeters or 
more, it is advisable, provided there is no contraindication, to 
obtain the specimen by the use of wet cups. It must be borne 
in mind that by this method there is always more or less ad- 
mixture of lymph. Another method is to draw the blood from 
a dilated vein into a large sterile antitoxin syringe. Lastly, an 
ordinary venesection may be resorted to. 

The withdrawal of blood, if aseptically performed, is prac- 
tically free from danger and need disturb the patient very little. 
Before proceeding in any case it is advisable to determine the 
absence of hemophilia in the patient. 



ESTIMATION OF THE HEMOGLOBIN. 



51 



ESTIMATION OF THE HEMOGLOBIN. 

Method of Gower. — Gower^s hemoglobinometer consists 
of: (a) Two glass tubes, one of which contains a standard solu- 
tion of picrocamiine in glycerin jelly of a color equal to a 1 per 
cent, solution of normal blood. The other tube is graduated in 
percentage up to 120. (h) A graduated capillary pipette meas- 
uring 20 cubic millimeters with a rubber filling tube attached. 




Fig. 10. — Fleischl Hemoglobinometer, surrounded by accessories 
necessary to performance of the test, 

INCLUDING DALAND LANCET. 



(c) A long, fine glass pipette with rubber bulb for diluting the 
blood with distilled water. 

The standard and graduated tubes are flattened on two 
sides, and for comparison are placed in a rubber stand. A white 
light should be used, which should be made to shine through a 
sheet of white paper during the comparison of the colors. 

Tbchnic. — Twenty cubic millimeters of blood are sucked 
up into the graduated pipette and blown into the graduated 
tube; distilled water is then drawn up to the same mark and 
blown into the graduated tube; by this means the capillary 
pipette is cleaned and no hemoglobin lost. Then by means of 



52 CLINICAL HEMATOLOGY. 

the other pipette, distilled water is added, drop by drop, to 
the blood, shaking it frequently, until the color corresponds with 
that of the standard. The number on the graduated scale at the 
level of the diluted blood, when the two tubes are of the same 
tint, represents the percentage of hemoglobin. 

This instrument is accurate between 2 and 3 per cent., but 
below a reading of 10 per cent, it is very difficult to judge with 
certainty any difference in the tint. This may be overcome by 
employing two or more pipettes full of blood and then dividing 
the results by the number of pipettes (20 cubic millimeters 
each) employed. 

Method of Fleischl. — The instrument consists of a metal 
stand having a horizontal table with a circular aperture in its 
center, beneath which is placed a reflector of plaster-of-Paris. 
Immediately beneath the aperture and above the reflector, a 
graduated wedge of tinted glass is arranged to move in a hori- 
zontal plane by means of a milled thumbscrew. This graduated 
wedge of glass is shaded to represent varying degrees of hemo- 
globin content, and carries with it a scale indicating the blood- 
strengths. For collecting and diluting the blood for examina- 
tion, a pipette and diluting chamber are furnished. llie 
technic is as follows : — 

By the aid of the pipette which is furnished with the ap- 
paratus, an exactly determined amount of blood is dissolved in 
a measured quantity of distilled water (the contents of one-half 
of the diluting chamber, the other half being filled with an 
equal amount of distilled water) . 

As the capillary pipettes vary in. size for each chamber 
accompanying apparatus, it is essential, when purchasing new 
pipettes, to obtain those having a number on the metal handle 
the same as on those originally accompanying the instrument; 
otherwise, the observation will be incorrect. The cell thus pre- 
pared is then placed over the aperture in the table, and artificial 
(candle, lamp, or gas) light is directed through it by means 
of the reflector. The part of the wedge is now searched for 
which accurately compares in tint with the solution of blood 
under examination, and the number of the scale is read ofl that 
corresponds to this point on the glass wedge. (It is a matter 
of common experience that the grading of these instruments is 



ESTIMATION OF THE HEMOGLOBIN. 



53 



too high, and that a specimen of blood that corresponds to 90 or 
95 on the scale is normal.) 

Hemoglobinombtbr op Fleisciil-Meischer. — A recent 
improvement in the Fleischl hemoglobinometer has been made by 
Meischer. This instrument (Fig. 11) is similar in general 
appearance to the original Fleischl. Tt has the same stand and 
the same scale principle, although this latter is standardized 




Fig. 11.— Fleischl-Meischer Hemoglobinometer. 



differently and graduated on a different basis. It differs 
materially in the method of measuring and diluting the blood, 
in the form of the comparison chamber, and in the meaning of 
the graduation of the scale. 

The diluting pipette is similar in construction to the pipette 
of the hemocytometer, its calibrations, however, being diffoi-- 
ent. The marks are i/o, %, and 1. Above and before each 
of these main divisions are two marks, each correspondino- to 
Moo of the contents of the capillary {ube. This device enables 
the worker to measure accurately the column of blood taken. 
in case he gets too little or too much blood in the tube. The 



54 



CLINICAL HEJVIATGLGGY. 



relation of the capillar}^ to the ampulla is such that blood, 
drawn to the mark 1 and diluted to the mark above the am- 
pulla, gives a dilution of 200; if drawn to the mark %, the 
dilution is 300, while the line Y2 fnrnishes a dilution of 400. 
The diluent used is %o P^r cent, sodium carbonate solution. 
This dissolves the stromata of the red cells and produces a 
clear solution. If the diluent becomes turbid after standing 
some time, it should be fieshl}^ made and should contain no 
bicarbonate. 

Calculation of Eesults. — ^This is possible only with the 
use of the "table of calibrations," which contains the scries of 




Fig. 12.— Sahli's Hemoglobinometer. 



scale divisions and the absolute amount of hemoglobin in milli- 
grams per 1000 cubic centimeters of blood, corresponding to 
each division of the scale when the 15-millimeter chamber is 
used. 

The principle of Sahli's hemogiobinometer (Fig. 12) is 
taken from Gower's hemogiobinometer, but contains a more 
permanent standard solution. The latter is made by the 
action of hydrochloric acid up6n blood, so that hydrochlorate of 
hematin is produced, which serves as the standard color. In 
tint it corresponds to a 1 per cent, solution of normal blood. 
The blood to be examined is also acted upon with a definite solu- 
tion of hydrochloric acid (Yio normal) and the color altered 
by reason of a similar formation of hydrochlorate of hematin 
varying in intensity according to its hemoglobin content. Thus 



ESTIMATION OF THE HEMOGLOBIN. 



55 



two solutions of the same color are compared. The blood solu- 
tion is mixed in a glass tube graduated to represent percentages 
of hemoglobin. Twenty cubic millimeters of blood are drawn 
into a pipette similar to that of Gower^s; this is blown into the 
graduated tube, which has been filled to the mark 10 with the 
hydrochloric acid solution. When the mixture has become a 
clear dark-brown color, distilled water is added until it cor- 
responds to the standard color solution, and the percentage of 
hemoglobin is read off on the scale. The two tubes are fitted 
into a black vulcanite holder which acts as a light screen and 





Fig. 13.— Dare's Hemoglobinometer. 
A, With Candle Light; B, With Electric Light. 

which is backed by a ground-glass plate for a light diffuser. 
The comparison is made by transmitted light, the result being 
the same whether it be made in artificial or in day light. 

Method of Dare. — This instrument (Fig. 13), introduced 
by Dare, has the advantage of using undiluted blood; hence it 
avoids any error consequent upon dilution. The principle of 
this instrument is as follows: The color of undiluted blood is 
compared by artificial light with that of a graduated glass scale 
made of ruby glass (purple of Cassius) the 100 point of which 
is standardized against a solution of 13.77 grams of hemoglobin 
in 100 cubic centimeters of serum. 

Method. — Swing outward the movable screen, which serves 
as a cover for the case; adjust the eye-tube, and fit the candle 
attachment in its place opposite this. The candle should be 
so adjusted that its upper end is flush with the top of the clasp 



56 CLINICAL HEMATOLOGY. 

which holds it. See that the pipette^ ■composed of the rectan- 
gular glass plates, is thorougly cleansed and dry. The space 
between the plates is filled by applying the edge of the pipette 
to the side of a fairly large drop of blood. Adjust this pipette 
in its place and rotate the color scale, by means of the milled 
screw, until the colors match. Hold the instrument steady to 
prevent any flickering of the flame. 'No dark room is necessary, 
but it is advisable to point the instrument at some dark object 
and to avoid direct sunlight, as the shadings of color are not 
so easily matched by direct daylight. As soon as the colors are 
matched make the readings. This reading is observed on the 
left side of the case in the small open space, — the line which 
coincides with the beveled edge of the opening represents the 
percentage of hemoglobin, on the basis of a value of 13.77 
grams of hemoglobin per 100 cubic centimeters as 100 per cent. 
It is, therefore, easy to calculate the direct amount of hemo- 
globin in the blood examination. 

This instrument has the advantages that undiluted blood 
is used, that the scale of comparison is usually very accurately 
standardized, that it is convenient, easy of manipulation, and 
rapid in giving results. Coagulation of the blood does not occur 
sufficiently soon to introduce an error, providing the reading is 
taken within a reasonable time. It is more convenient for gen- 
eral use than most others, can be used in a light room, but it 
is rather expensive, and an occosional inaccurate instrument 
has been found. 

ENUMERATION OF THE CORPUSCLES. 

Description of Apparatus. — ^In the estimation of the 
blood corpuscles and the blood platelets a measured portion of 
blood is drawn into a suitable mixing pipette, diluted a defi- 
nite number of times and placed upon an especially ruled 
slide known as a counting-chamber; a certain number of cells 
are then counted in a measured portion of the field, from which 
the number of cells in 1 cubic centimeter of undiluted blood 
are computed. 

The pipettes which are used in collecting, diluting and 
mixing the blood specimen are known as "melangeurs." The 
pipette used to measure and dilute the blood for the red-cell 



ENUMERATION OF THE CORPUSCLES. 57 

count has a fine capillary bore expanded into a bulbous por- 
tion near the upper end, in which is confined a small glass bead 
for the purpose of obtaining a uniform suspension of cells in 
the diluting fluid. Thd capillary portion of the tube is grad- 
uated equally into ten divisions, while above the mixing 
chamber is another mark. The fifth graduation is marked 0.5, 
the tenth 1.0 and the upper one 101. These marks serve to 
indicate the dilution and have the following significance. If 
the fresh blood is drawn to the 0.5 mark and the diluting fluid 
to the 101 mark, then the dilution of blood in the mixing 




Fig. 14.— Levy-Neubauer Hemocytometer. 

chamber will be one to two hundred, whereas if the blood is 
drawn to the 1.0 mark, then the dilution will be one to one 
hundred (this latter dilution is only employed in the examina- 
tion of specimens known to have relatively few cells per cubic 
centimeter). 

The white-cell pipette is similar in construction to the red 
cell pipette except that itj is of larger bore and the markings 
are 0.5, 1.0 and 11 respectiveh', indicating dilutions of one 
to twenty and one to ten. In preparing specimens for blood 
count it is customary to employ difl^erent diluting fluids for 
the red and white cells. By so doing it is possible to destroy 
the cells not under consideration, thereby facilitating the 
count. Thus a solution of 2% per cent, potassium bichromate 
preserves the red-cells and destroys the white-cells, while a ^^2 



58 



CLINICAL HEjMATOLOGY. 



per cent. solTition of acetic acid (tinted with a basic stain) 
will preserve the white^ while rendering the red cells invisible 
(see Appendix for other diluting fluids). 

Description of Cowiting Chamber. — The original Thoma 
counting chamber was so constructed that the floor of the 
chamber with the cover-glass in place was exactly %o n^iHi- 
meter below the under surface of the cover-glass. In the 
center of this depression, the surface of the slide was etched 
by fine microscopic lines into minute squares of 34o niilli- 
meter on each side or ^oo square millimeter. The application 
of the cover-glass producing minute cubic divisions having 
a unit capacity of %ooo cubic millimeter. The slide pos- 
sessed 400 small squares divided by additional lines into 16 



[] r 

^mmieep 




If 

Si: m 


1 
1 

T 


! PHILADELPHIA. 

! j.s.Fw:;.TiJo, liKcsi. 



Fig. 15.— Counting Chamber with Single Nedbauer Ruling. 



groups of 16 small squares each, the total of which was 256 
small squares. This counting chamber was used in the estima- 
tion of both red and white cells, also for counting the number 
of cells in the cerebro-spinal fluid and other body fluids. The 
new counting chamber with the ruling of I^eubauer is a dis- 
tinct improvement over the earlier types in that it eliminates 
the element of error resulting from undue pressure upon the 
cover-glass in chambers of the closed type. 

The N'eubauer single ruling counting-chamber (see Fig. 
15) is now universally used. It is of the '^^open type" with 
central ruling similar to the original Thoma in so far as the 
cubic capacity of the small squares is concerned. It differs in 
that beside the central ruling above described, there are eight 
additional large squares formed by extension of the lines 
forming the small squares together with additional lines ex- 
tending to and forming the eight additional large squares, 
each of which are divided into 16 small S(^uaxes^ each large 



ENUMERATION OF THE CORPUSCLES. 59 

square representing %o cubic millimeter when the counting 
chamber is covered (see Fig. 17). 

Estimation of Red Blood Cells. — The ''Melangeur" having 
the smallest bore and having marks upon it at 0.5, 1.0, 101 is 
used for this estimation. The drop of blood issuing from the 
puncture is drawn up to the 0.5 mark and the tip then quickly 
freed from adherent blood and immersed in the diluting fluid 
which should be immediately drawn up to the 101 mark. The 
tip of the tube is then stopped with the finger, to prevent escape 
of fluid, and vigorously shal^en for at least a, minute, to insure 
thorough and even dilution of the blood. Before filling the 
chamber the diluting fluid contained in the capillary part of the 
tube is blown out and wiped away. The tip of the pipette is then 
placed at the edge of the center ridge of the counting cham- 
ber and the solution allowed to run by capillarity under the 




Fig. 16.— Daland Hematokrit, showing one percentage 
tube in position. (a. h. t. co. ) 

cover-glass, which should previously have been placed into 
position. The count should not be commenced until a few 
moments have elapsed, for the cells to settle. 

A simple and practical method of arriving at the number 
of red corpuscles obtained in a cubic millimeter of blood is the 
following : Select and count from the various parts of the cham- 
ber fivq large squares (Fig. 17), counting the border cells only 
upon the upper and right-hand lines. The total of small 
squares counted will be eighty. To the total of the corpuscles 
counted in the five large squares add four 0000, and the number 
resulting will be the number of red corpuscles in 1 cubic milli- 
meter of undiluted blood. 

Enumeration of the Red Cells. — Explanatory note: Y^qoo cubic 
millimeter equals the cubic capacity of one small square, i/^oo equals 
the dilution of the specimen of blood. Five large or SO small 
squares are the number of squares counted. Then the number of cells 
per cubic millimeter in the undiluted specimen -will equal the number 
of cells in one small square multiplied by the dilution times 4000, viz.: 



60 CLINICAL HEMATOLOGY. 

— Let X equal cells per cubic millimeter, and let y equal number of red 
blood-cells in 80 small squares. Then, /_ X 200 X 4000 = x, which, 
simplified, is the same as y X 10,000 = x = the number of red blood- 
cells in one cubic millimeter of undiluted blood. 

Determination of the Erythrocytes by the Centrifuge. — 

The Dalaiid hematokrit (Fig. 16) offers a quick, simple, and 
accurate method of determining the number of red corpuscles 
in the blood. The two great advantages of this process are, 
first, the elimination of the personal equation, and, second, as 
there is no dilution of the blood required, the frequent source 
of error arising from this cause does not enter in. 

The hematokrit consists of an extremely light, though very 
strong, metal frame containing two glass tubes, each 50 milli- 
meters long and graduated in one hundred parts, and having 
a uniform lumen of 0.5 millimeter. 

The length of the percentage tubes and their distance from 
the center of the revolving hematokrit frame must always be 
the same as was used in the original experiments by which the 
comparative determinations were made, since any variations in 
these factors will produce corresponding variations in the 
results obtained. 

The Technic. — The blood having been secured as out- 
lined on page 50, the blunt end of the graduated percentage 
tube is attached to a suction pipette and the tube completely 
filled with freshly drawn blood. After any excess of blood has 
been carefully wiped away, the blunt end of the tube is placed 
in the distal cap of the hematokrit frame, and the other end 
pressed down upon the inclined plane of the proximal cap until 
it falls into and locks in the cavity. The second tube should 
be similarly treated and placed in the opposite side to serve as 
a check. It is absolutely necessary that the entire procedure 
should be performed with celerity so as to anticipate coagula- 
tion. After filling, immediately place the frame upon the spin- 
dle of the high-speed centrifuge, and turn the handle at the uni- 
form rate of eighty turns per minute for exactly two minutes. 
This will produce 10,000 revolutions per minute in the spindle. 
At the expiration of this time all the corpuscles will be found 
to occupy the distal end of the tube; a thin, almost invisible, 
whitish line of white corpuscles appears between them and the 



ENUMERATION OF THE CORPUSCLES. 61 

plasma, which will be found to occupy the proximal portion of 
the tube. With the aid of a hand lens the height of the column 
of red corpuscles is read off, and the reading multiplied by 100,- 
000, which converts the volume percentage into the number of 
corpuscles per cubic millimeter of the blood under examination. 

The speed of the handle of the centrifuge must be uniform 
throughout the whole period, because this rate of rotation is 
the measure of a definite amount of centrifugal force gener- 
ated in the revolving arms of the hematokrit frame. If the 
rate of rotation is either increased or decreased, the height of 
the column of cells will be similarly affected, thereby producing 
either too low or too high a reading. 

A possible source of uncontrollable error is the variation 
in size of the individual erythrocytes which occurs in certain 
pathologic states. The most accurate results by this method 
will, therefore, be obtained when the corpu.scles are of uniform 
size. This fact has been taken advantage of in the estimation 
of the volumetric quotient, and its relation to diagnosis and 
prognosis in certain blood conditions (see below). 

The Percentage of Erythrocytes. — To obtain the percent- 
age of red blood-corpuscles, take the first two left-hand figures 
of the count in 1 cubic millimeter and multiply these by two; 
this will represent, approximately, the percentage of red blood- 
cells. 

Explanation". — If we consider normal blood to contain 
exactly 5,000,000 red corpuscles, then the figure 50 x 2 would 
equal 100 per cent., or normal. If, however, only 4,790,000 were 
counted in 1 cubic millimeter of blood, then 4-7 x 2 will equal 
94 per cent, of erythrocytes. 

The Color-Index. — From a knowledge of the percentage 
of hemoglobin and the percentage of cells, we are in a position 
to calculate the average color-index per cell, i.e., its relative 
hemoglobin-content as compared with the normal cell. 

Example. — If the percentage of hemoglobin be 100 and 
the percentage of cells 100, then the color-index of each cell 
would be 1.0 or normal, viz. : — 

100 % hemoglobin 

= color-index 1.0. 

100 % red cells 



62 CLIiSTICAL HEMATOLOGY. 

If the percentage of hemoglobin be 67, and the percentage 
of corpuscles 90, then each individual cell will contain less than 
a normal amomit of hemoglobin : — 

67 % hemoglobin 

= color-index 0.74. 

90 % red cells 

The Volumetric Quotient or the Volume-Index of the 
Erythrocytes. — The investigations of Capps^ have demonstrated 
that the normal volume obtained by the centrifuge hematokrit 
is 50 per cent, in the normal specimen. This volume he desig- 
nates as 1. The volume in pathologic alterations can be cal- 
culated in percentage of the normal volume, just as is the 
amount of hemoglobin estimated under similar circumstances. 
Having determined the volume by the hematokrit the erythro- 
c}i:es are next counted and their number expressed in percent- 
age by comparing with the normal. By dividing the volume of 
erythrocytes by the number of erythrocytes in the same blood 
(both expressed in percentage), we obtain the "volume-index^^ or 
"volume-value" of the erythrocytes, which is analogous to the 
color-index of hemoglobin as obtained above. This number in a 
measure of the average volume of the individual erythrocyte, 
and obviously under normal conditions, equals 1. Capps found 
that an increase in the volume-index of the erythrocytes is one 
of the most constant and accurate characteristics of pernicious 
anemia, which agrees with the well-known fact that many 
macrocytes appear in the blood in this disease, and that the 
color-index is also greater than one. In contrast to this, the 
so-called secondary anemias usually show a diminished volume- 
index ; the same is true of chlorosis, in which aSection the 
volume-index may be taken into account in determining the 
prognosis, since a normal or only slightly decreased volume- 
index gives a favorable prognosis, whereas a markedly dimin- 
ished volume-index may be considered a bad prognostic sign. 

When utilized in this way the volume-index may become 
a more reliable sign in chlorosis than the percentage of hemo- 
globin or the color-index.^ 



2 Jour. Med. Research, vol. x, 3, Boston, 1903. 
* Sahli's "Diagnosis," quoting Capps : loc. cit. 



ENUMERATION OF THE CORPUSCLES. 



63 



Estimation of Leukocytes. — Selecting the pipette of larger 
caliber which provides a dilution of 1 to 10 or 1 to 20, a fresh 
drop of blood is drawn up to the 0.5 mark, free from exces- 
sive blood about the top and the proper diluting fluid 
promptly drawn up to the 11 mark. Mixing and preparing 
the slide is done in the same manner as estimating the ery- 
throcytes (see page 59). 

In order to accurately count the white cells, it is neces- 
sary, because of their relative minority, to> count only large 
squares. This is usually done in the following manner: use 
the low power (%) objective which brings a large square in 
view, ignore the small squares and commence in the upper 




Fig. 17.— Detail of Nedbauer ruling. 



right hand corner to count 9 large squares (that is, all cells 
within ruling), divide the number of cells counted by nine 
and multiply by 300. If during dilution the blood is drawn 
up to the 1 mark, then the multiplier is 100. For example : — 

Total cells counted 298 

Total squares oounted 9 

298 divided by 9 equals 33.1 

33.1 times 200 (dilution 1:20) equals 6620. cells 
per c.c. 

It is not always necessary to adhere to this formula since 
clinical accuracy is maintained when a total of 250 white cells 
have been counted, therefore, the usual practice is to com- 
mence in the right hand upper corner and continue counting 
white cells until not less than 250 have been counted, noting 
the number of large squares covered to reach this number. If 
this number is reached before a large square is completed, finish 



64 CLINICAL HEMATOLOGY. 

coimting that square. Divide this number by the number of 
large squares counted and multiply by 200 ; for example : — 

Total cells counted 316 

Total squares counted '. . . 5 

316 divided by 5 equals 66.6 

66.6 times 200 equals 13,320 cells per c.c. 

CLEANING BLOOD TUBES AND PIPETTES. 

Pipettes washed several times with water, followed inmie- 
diately with alcohol and then ether, rarely become clouded. If 
any coagulum forms, 10 or 20 per cent, antiformin will speedily 
remove it. 

Nitric acid should not be used for cleaning the tubes, be- 
cause it tends to form a coagiilum within the tube which is hard 
to remove. If any coagulum forms, caustic soda or caustic 
potash will remove it. 

The Counting Diaphragm. — The process of counting may 
be simplified by using a diaphragm over the eyepiece, which 
has a central square aperature, the area of which, with the 
same lens, eyepiece, and length of draw-tube, exactly corres- 
ponds to the area of the magnified 400 small squares, i.e., %o 
cubic millimeter. With the covered chamber any area of it 
viewed through this diaphragm is equivalent to %o cubic milli- 
meter, so that after being once adjusted any part of the field 
may be counted^ without taking any note of the ruled lines. A 
movable stage is a great convenience. These diaphragms may be 
obtained with the instnmient, or easily made from a piece of 
cardboard or thin metal. 

COUNTING THE BLOOD-PLATELETS. 

These bodies are round, oval or rod shaped, 3 ^ in diam- 
eter, bluish in color, homogeneous, rarely granular, and stain 
lightly with both acid and basic dyes. They have no nucleus 
and have a decided tendency to clump when shed. 

The method of obtaining blood to count the platelets is 
the same as that for blood. 

Specimens of platelets are best obtained by puncture of the 
finger or ear-lobe, directly through a drop of a 10 per cent, 
solution of sodium metaphosphate. 



MICROSCOPIC EXAMINATION. 65 

The technic of counting these cellular elements has, until 
recently, been imperfect and the results variable. In 1911 
Wright and Kinnicutt^ introduced a method which is simple, 
exact, and fairly reliable. The technic is as follows: The 
blood is diluted 1 : 1000 by means of the pipette used for 
counting the red cells, and the counting is done in the Thoma- 
Zeiss counting chamber, using all the precautions previously 
mentioned. The specially thin cover-glass of Zeiss, with central 
excavation, is used to render the platelets clearly visible. The 
diluting fluid consists of 2 parts of a 1 : 300 aqueous solution 
of 'H^rilliant cresyl blue" and 3 parts of a 1 : 400 aqueous solu- 
tion of potassium cyanid. These two solutions should be fairly 
fresh, kept separate, to be mixed and filtered just before taking 
the blood. After the counting-chamber has been filled, ten to 
fifteen minutes should elapse before counting, in order to allow 
the blood-platelets to settle to the bottom of the chamber and 
so be more easily and accurately counted. The platelets appear 
as sharply outlined, round, oval, or elongated, lilac-colored 
bodies, some of which form a part of small spheres or globules 
of hyaline substance. The red cells are decolorized and appear 
as "shadows," while the nuclei of the leukocytes are stained a 
dark blue and their protoplasm light blue. This method shows a 
normal platelet count of 2.25,000 to 350,000 per cubic millimeter. 
No constant relations seem to obtain between the variations 
in the numbers of platelets and of the leukocytes. Accord- 
ing to Determann, the ratio between the red cells and the blood- 
platelets is, on the average, 22 : 1. 

MICROSCOPIC EXAMINATION. 

Examination of a Drop of Fresh Blood. — ^If a drop of fresh 
blood is placed upon a slide and a perfectly clean cover-glass 
allowed to fall upon this, a fresh preparation for examination 
will be produced, which may either be examined immediately 
or, if sealed around the edge with a little vaseline, may be car- 
ried to the laboratory and examined within an hour. With the 
aid of a 6 objective many interesting points may be observed. 
First, the shape of the red cells and their hemoglobin staining; 

* Jour. Amer. Med. Assoc, vol. Ivi, 1911, p. 1457. 

5 



QQ CLINICAL HEMATOLOGY. 

also rouleau formation. Second, the structure of the white 
corpuscles by which the different varieties may be roughly dif- 
ferentiated. Third, the Uood-platehts. Fourth, a rough idea 
of the relative number of red and white cells may be formed. 
This matter will be discussed more in detail below. 

Studies of the minute structure of the leukocytes cannot 
be made satisfactorily by this simple method, as some of the 
forms are present only in small numbers, and are detected 
with difficulty. Further, prolonged examination of the fresh 
specimen allows time for changes, which rapidly obscure the 
identity of the cells; hence we are obliged to resort to a method 
of preparing a specimen which shall be permanent and which 
can be stained as a further aid to differentiation. These meth- 
ods have the added advantage that they allow the investigator 
to work quite independently of the presence of the patient, to 
choose the time and place of examination, and to permit at any 
time of verification and demonstration of the original result 
obtained. 

Prepaeation" of the Specimen". — The blood is obtained 
from the tip of the finger or the lobe of the ear after the man- 
ner just described. Cover-glasses should previously be pre- 
pared for reception of the fresh blood as soon as it is drawn. 
The cover-glasses should be square, seven-eighths or one inch 
in diameter, and of special thinness (0.1 to 0.08 millimeter). 
They should be washed carefully with warm water and soap, 
dried with fat-free gauze, and finally wiped off with ether. This 
is necessary to insure proper spreading of the blood between the 
two glass surfaces, which would be prevented by traces of fat 
or particles of fiber, dust, etc. Just before the glasses are 
used they should be wiped off with a piece of silk or tissue 
paper, and thereafter handled with care by the corners, or, 
better still, with forceps. This preparation of the cover-glasses 
may be done in the laboratory or carried on at the bedside, as 
desired. 

The glasses being ready and the finger cleansed and punc- 
tured, a small drop of blood is allowed to exude without pres- 
sure. Now a cover-glass is taken up diagonally between the 
thumb and finger of one hand, and its center allowed to touch 
the drop of blood. Immediately taking another cover-glass in 



MICROSCOPIC EXAMINATION. 67 

the same manner in the other hand, it is allowed to fall upon 
the drop. If the glasses are perfectly clean and the maneuver 
properly executed, the blood will immediately spread out in a 
uniform layer between the two glasses. The two cover-glasses 
are now separated one from the other by a rapid sliding motion, 
in the strict plane of their surfaces, without the slightest lifting 
or tilting. 

If the drop taken was small enough and the technic prop- 
erly carried out in every detail, the result will be two uniform 
smears, covering the larger portion of the surfaces of the cover- 
glasses, which, when examined under the microscope, will present 
a uniform layer of corpuscles, with little if any overlapping of 
the individual cells. 

Usually six films are prepared in this manner and allowed 
to dry in the air without heat. 

Methods of Fixation. — In using the Romanowski stains 
no previous fixation of the albuminous film is required. 'Before 
applying any of the other stains it is necessary to "fix^^ the 
blood-film. If this is not done the corpuscles will be washed 
from the glass by the fluids applied. 

1. A strip of sheet copper eighteen inches long and two 
or three inches wide is placed upon a stand, and the flame from 
a Bunsen burner placed under one end. After allowing suf- 
ficient time for the plate to attain a maximum heat the films 
are placed upon it at a point where a drop of cold water fails 
to roll off, but adheres to the hot metal and steams away. At 
this point fixation is complete in from one-half to three-quarters 
of an hour. 

2. Fixation may be accomplished by immersion for five to 
ten minutes in a mixture of 1 part formaldehyde solution and 
99 parts absolute alcohol. 

3. For routine work when fair accuracy, accompanied by 
speed in obtaining results, is necessary, fixation in the naked 
flame of a Bunsen burner may be practised. This is accom- 
plished by holding the dried smear diagonally between the thumb 
and forefinger of one hand, film side up, and passing it through 
the flame a number (five to ten) of times with sufficient rapid- 
ity to prevent the fingers being burned. 



68 CLINICAL HEIVLVTOLOGY. 

Methods of Staining: Eosin and Methylene-Uue. — The 
fixed film held in the cover-glass forceps is flooded with a 
14 per cent, solution of eosin in 50 per cent alcohol, which is 
gently washed off with distilled water after the lapse of one or 
two minutes. The counter-stain of methylene-blue, 1 per cent, 
aqueous solution, is then added and allowed to act for from 
two to five minutes, according to the density of the film. 
Finally, washing in distilled water, blotting and drying, complete 
the process prior to mounting and examination. 

This is an extremely simple, yet effective and permanent, 
method of staining blood-smears. Little difficulty will be ex- 
perienced in obtaining satisfactory specimens. The eosin should 
not be allowed to act too long, but there is little danger of over- 
staining with the methyiene-blue. Both of these stains are 
permanent when prepared, and can be kept until exhausted. 

Properly performed, this method gives very vivid and con- 
trasting pictures, the nuclei of the different white cells tak- 
ing various shades of blue, pale in the lymphocytes, and dark 
in the polymorphonuclears. The protoplasm appears faintly 
tinted blue (paler than the nuclei). The eosinophilic granules 
are a bright pink ; the erythrocytes a dusky red. 

Ehrlich's Triacid Staining Method (for formula and prepa- 
ration see Appendix). — The smear is taken in the cover-glass 
forceps and a few drops of the prepared stain placed upon it 
and allowed to remain for about five minutes. There is little 
danger of overstaining. The specimen is next washed in dis- 
tilled water and thoroughly dried prior to mounting. If 
mounted in Canada balsam it is imperative that this should 
not contain any chloroform; otherwise the colors will gradually 
become blurred. 

The Romanowslci or ''Universal Staining Method. — Leish- 
mann's modification of the Eomanowski stain, as made by 
Wright 5 (see Appendix for stain), is the stain used. This 
method is altogether quicker and easier than the method of 
Ehrlich. It requires no fixing fluid or heating apparatus, and 
gives pictures which are uniformly superior. 

TecJinic. — Allow three or four drops of the prepared stain 
to fall upon the smear and permit it to remain one-half min- 
ts Wright : Jour. Med. Research, vol. vii, 1902. 




Normal and Pathological Blood-cells. 

7, Normal red cell, or erythrocyte ; diameter, 7.5 ^^.. 2, Nucleated 
red cell, or normoblast ; diameter, 7.5 m. 3, Megaloblast ; diameter, 8 
to 12 /* ; seen in pernicious anemia. 4, Small lymphocyte ; diameter, 6 
to 8 At ; average in normal blood, 20 to 26%. 5, Large lymphocyte ; 
diameter, 8 to 13 /* ; average in normal blood, 5 to 9 %. 6, Polymor- 
phonuclear leukocyte; diameter, 10 to 11 fi', average in normal blood, 
65 to 70%, 7, Eosinophile ; diameter. 19 to 12 m : average in normal 
blood, Yi to 2%. 8, Large mononuclear leukocyte : diameter, 12 to IS 
/* ; average in normal blood, 1 to 2%, 9, Transitional : diameter. 12 
to 17 /* : average in normal blood. 2 to 3%. 10, Neutrophilic mvelocyte ; 
diameter, 12 to 20 fi; seen in myelogenous leukemia. 11, Eosinophilic 
myelocyte ; diameter, 10 to 16 iu ; seen in myelogenous leukemia. 



MICROSCOPIC EXAMINATION. 69 

ute, rocking the cover gently so as to insure an even distribu- 
tion of the stain. N"o attempt is made to check evaporation. 
At the end of one-half minute add double the quantity of dis- 
tilled water, i.e., six to eight drops, and allow it to mix with the 
alcoholic stain. Immediate mixing is hastened by gently rock- 
ing the cover-glass. The film is now allowed to stain for five 
minutes. In thick smears ten minutes may be necessary. The 
stain is now gently washed off with distilled water, and a few 
drops of water are allowed to rest on the film for one minute, 
when it should be dried in air and mounted. 

Appearanc*e of Stained Blood-films. — Erythrocytes, pale 
pink or greenish, semi-transparent. 

PolymorpJionuclears. — Nuclear network, stained a very 
ruby-red color, or with sharply defined margins. Extra-nuclear 
protoplasm, colorless. Fine eosinophilic granules, red. 

Mononuclears. — N'uclei ruby-red with extremely sharp, 
clear outlines. Extra-nuclear protoplasm, pale eau-de-nid or 
blue, occasionally showing a few red granules. 

Lymphocytes, — Same as mononuclears, except that as a 
rule the nuclei are more deeply stained. 

Coarse-grained Eosinophiles. — Nucleus only, red, but not 
so densely stained. Granules pale pink. 

Basophiles. — Granules very densely stained of a very pur- 
plish black tint. Nucleus red, but usually more or less meshed 
by granules overlaying it. 

Nucleated Bed Cells. — Nucleus almost black, with sharp 
outline, extra-nuclear portion gray. 

Blood-plates. — Deep ruby-red with spiky margins, fre- 
quently showing a pale-blue spherical zone surrounding the red 
center. 

Bacilli and Micrococci. — ^Speaking generally, these stain 
evenly blue, but by prolonging the staining period aad subse- 
quently decolorizing with absolute alcohol many interesting 
variations may be noted with different organisms by which 
structural details are brought out not generally observed by 
other staining methods. 

Malarial Parasites. — ^The body of the parasite stains blue 
and its chromatin ruby-red. In the case of the tertian parasite. 



70 . CLINICAL HE^IATOLOGY. 

Schuffner's dots are well marked in the containing red blood- 
corpuscles. 

The only weak points in the Eomanowski stain are said 
to be the deceptive resemblance between megaloblasts, certain 
lymphocytes and certain myelocytes, and failure to differentiate 
the basophiles. 

Eosin-hematoxylin. — This stain is especially important in 
cases in which the nuclear structures are to be studied. It 
stains the nuclei beautifully, showing their fine structure, 
karyokinetic figures, and pycnotic qualities, as well as the baso- 
phile granules of both red and white cells (for preparation of 
reagents see Appendix, page 480). 

Technic. — Stain the specimen with the eosin solution for 
cne-half minute, and wash in water. Without drying place the 
slide in the hematoxylin solution for one to three minutes, the 
time varying with the particular stain and with the experience 
of the worker. Wash with water, dry, and mount. 

TERMS IN COMMON USE IN CLINICAL HEMATOLOGY. 

Anemia. — A condition of the blood in which there is a 
deficiency in one or more of the normal constituents. 

Anhydremia. — A deficiency in the normal fluid of the blood. 

Basophilic Granulation. — A peculiar granular degeneration 
of the red blood-cells which is noted in chronic lead-poisoning. 

Hydremia. — An excess of fluid in the blood. 

Hyperglycemia. — Excess of sugar in the blood. 

Leukocytosis. — An increase above the normal number of 
white blood-cells. 

Leukopenia. — A diminution in the number of white blood- 
cells. 

Lipemia. — The presence of an abnormal amount of free 
fat in the blood. 

Macroblasts. — Nucleated red blood-cells of more than nor- 
mal diameter. 

Macrocytes. — Abnormally large red blood-cells. 

Melanemia. — ^The presence of free pigment in the blood. 

Microblasts. — Nucleated red blood-cells of abnormally 
small size. 



VARIETIES OF LEUKOCYTES. 71 

Microcytes. — Abnormally small red blood-cells. 

Normoblasts. — Nucleated red blood-cells of normal diameter. 

Oligemia. — By this term is meant a reduction in the total 
volume of blood, both as regards the liquid and the cellular 
portions. 

Oligochromemia. — A diminution in the normal amount of 
hemoglobin. This may occur either independently or coin- 
cidently with a diminution in the number of red blood-cells. 

Oligocythemia. — A diminution in the number of red blood- 
cells. 

Plethora. — An increase in the total quantity of blood above 
normal. 

Poikilocytosis. — This term is applied to the very irregular 
shape of the erythrocytes observed in certain pathologic condi- 
tions. 

Polychromatophilic Degeneration or Anemic Degeneration 
(Ehrlich). — This is an atypical staining reaction of the eryth- 
rocytes, the significance of which is not yet definitely deter- 
mined. 

Polycythemia.— An increase in the number of red blood- 
cells as compared with the fluid-content. 

VARIETIES OF LEUKOCYTES. 

1. Normal Leukocytes. — (a) Lymphocytes: These are 
derived from the lymph-glands, and appear as small, round cells 
about the size of a red blood-corpuscle, with a large, centrally 
located nucleus, and a small margin of protoplasm. The nu- 
cleus stains rather intensely with the nuclear stains (hematox- 
ylin, methylene-blue, and Ehrlich's triple stain). The proto- 
plasm is free from granules. 

(b) Large Mononuclear Leukocytes. — ^These cells are 
two or three times as large as a red blood-cell, with a large, 
usually oval nucleus which is generally eccentrically placed. 
They stain poorly with nuclear stains. There is a relatively large 
amount of protoplasm, which is free from granules. They are 
derived from the bone-marrow, and may be regarded as the 
parent type of the polymorphonuclear. 

(c) Polynuclear or PolymorphonuclK'\.r (Neutro- 
philic) Leukocytes. — These are recognized by their multiple, 



72 CLINICAL HEMATOLOGY. 

irregular-shaped or bent nucleus. The nuclei stain very in- 
tensely, and the protoplasm is densely packed with neutrophilic 
granules. (See page 75 for Ameth's classification.) 

(d) Eosinophilic Cells. — These resemble the polymor- 
phonuclear cells, except that the small neutrophilic granules are 
replaced by coarse acidophilic (eosinophilic) granules. These 
granules are highly refractive, so that they can be readily recog- 
nized without staining. 

(e) Mast Cells. — ^These are cells of the polymorpho- 
nuclear type with marked basophilic granules, which are quite 
large, uneven, and irregularly distributed. They are not dis- 
tinctly stained by the triple stain. 

2. Pathologic Leukocytes. — (a) Mononuclear E'eutro- 
PHiLic Cells (Myelocytes) : These are large cells with a 
large, faintly staining nucleus, differing from the large mono- 
nuclear cells of normal blood by the presence of neutrophilic 
granules in the protoplasm. 

(b) Mononuclear Eosinophilic Cells (Ehrlich's 
Eosinophilic Myelocytes) : These cells are what their name 
implies, mononuclear eosinophiles. Very small cells of this type 
have been termed eosinopliilic microcytes. 

THE DIFFERENTIAL COUNT. 

The distinguishing characteristics of the various white 
cells, as brought out by any one of the differential stains above 
outlined, allow the investigator to separate these into several 
groups, and thus to estimate their relative numbers, which are 
usually expressed in percentage. The cells for a differential 
count can be enumerated without the aid of a mechanical stage, 
but this instrument of precision is a distinct aid in the per- 
formance of this procedure and should always be used when 
possible. Select a uniform and not too dense part of a stained 
smear, and adjust upon the mechanical stage so that the field 
presents cells in uniform arrangement without any overlapping. 
Now, by means of the thumb-screws of the mechanical stage, 
the field is carried back and forth before the eye of the observer, 
so that the same part of the field is not brought into view more 
than once (this matter is very simple when a mechanical stage 



THE DIFFERENTIAL COUNT. 



73 



is used) . As the parade of cells passes before the eye, the white 
cells are observed, classified, and the nnmber jotted down. This 
is continued until not less than two hundred (see Fig. 18, for 
computing chart to facilitate the count), and preferably five 
hundred or a thousand, cells have been counted. With the total 



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Fig. 18.— a Computing Chart to Facilitate Making a Differ- 
ential Leukocyte Estimation. (Devised by Dr. A. E. 
Osmond, Cincinnati, Ohio.) 

number of cells counted known, and also the number of cells 
in each class recorded, it is a simple matter to calculate the 
percentage of each variety of cell in the specimen examined. 

A Computing Chart for the Differential Leukocyte Count. — 
The accompanying chart (Fig. 18) has been suggested by Dr. 



74 CLINICAL HEMATOLOGY. 

Osmond^ to simplify the work and remove the sources of error 
in the differential count. The chart is inexpensive and can be 
made out of a piece of ground glass or of slate suitably ruled and 
marked in ink. The temporary markings are made in pencil 
and can be readily erased. It is figured out on a basis of two 
hundred cells and the marks on the left indicate the various 
types of cells encountered. At the bottom are the calculated 
percentages and at the top the actual number of cells counted 
when the vertical columns are filled. 

The heavy lines running vertically facilitate the count by 
indicating when fifty, one hundred or one hundred and fifty 
units are counted for a certain type of cell. By referring to the 
figures in the top row one can readily read the actual number of 
cells of each type counted and easily sum up when the total of 
two hundred has been reached. This being done, the percentages 
can be read off directly from the bottom figures, no calculation to 
determine these being required. 

The Normal Differential Count. — The normal leukocytic 
count may vary between 5000 and 10,000 white cells per cubic 
millimeter. The average normal leukocyte count is usually 
placed at 7500 leukoc3i;es per cubic millimeter. 

The minimum and maximum number of cells for each 
type, estimated from the minimum normal (5000) leukocyte 
count, is given in the following table: — 

VAEIETY. MINIMUM. MAXIMUM. 

Polymorphonuclears 3000 3500 

Small lymphocytes 1100 1500 

Large lymphocytes 250 350 

Eosinophiles 50 100 

Mast cells 5 25 

The minimum and maximum number of cells of each cell- 
type estimated from the maximum normal leukocyte count (10,- 
000) :— 

VAEIETY. MINIMUM. MAXIMUM. 

Polymorphonuclears 6000 8000 

Small lymphocytes 2200 3000 

Large lymphocytes 500 900 

Eosinophiles 100 200 

Mast cells 10 50 



6 A. E. Osmond : Jour. Amer. Med., Jan. 8, 1910. 



ARNETH'S CLASSIFICATION. 75 

For the average normal standard for each cell-type we may 
adopt the following standard: — 

VABIETY. NUMBEE. PEE CENT. 

Polymorphonuclears 4875 65 

Small lymphocytes 1950 26 

Large lymphocytes 525 7 

Eosinophiles 75 1 

Mast cells 8 0.1 

ARNETH'S CLASSIFICATION. 

Arneth,''' in his study of the blood both in health and dis- 
ease, has been led to classify the pol5Tnorphoniiclear neutrophiles 
into five classes, depending upon the number of nuclear lobes. 
His divisions seem to be fairly constant in health, but show 
great variation, especially in infectious conditions. Eeduced to 
tabular form, the Arneth classification appears on the following 



Clinical Significance. — It is probable that these various 
classes represent the gradual development of the polymorpho- 
nuclear neutrophile; the older the cells, the greater the tend- 
ency to reach Class 5, while in conditions associated with new 
and rapid cell formation, as in infectious conditions, we find 
the percentage of the earlier classes being increased, that of the 
later ones diminished. 

L. H. Briggs,^ in his investigation of the several types of 
neutrophile, found that the nuclear formula in the normal 
agrees closely and constantly with that found by Arneth and 
many other workers. In tuberculous, typhoid, pyogenic, and 
malarial infections a deviation from this normal is regularly 
found, the so-called ''shift to the left,'^ consisting of an increase 
in the cells with fewer nuclear units and a corresponding de- 
crease in the cells with many. The degree of this shift appears 
to be roughly proportioned to the severity of the infection, ex- 
cept in typhoid, in which it is uniformly present to a marked 
extent, irrespective of the individual case. In tuberculosis such 
changes in the neutrophiles seem to offer a distinct aid in prog- 
nosis, although its reliability needs furtlier confirmation. 



T T. Arneth : The Neutrophile Blood-picture in Infectious Diseases, Zeit. 
f. klin. Med., Ixvi, No. 2, 1908. 

8 California State Journal of Medicine, August, 1912, x. No. 8. 



76 



CLINICAL HEMATOLOGY. 





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LEUKOCYTOSIS. 77 

A Simple Method of Counting Eosinophile Leukocytes. — 

Dunger ^^ dilutes the blood in the proportion of 1 : 10 with the 
following solution: 1 per cent, aqueous solution of eosin and 
acetone, of each, 10 cubic centimeters; distilled water, 100 cubic 
centimeters. The eosinophiles can then be seen in a bright rose 
field as roundish spheres composed of shining red granules that 
can be easily recognized even by the inexperienced. They can 
then be counted much more readily than after preparations in 
the older ways. He considers that the eosinophiles form: a very 
sensitive indicator in the various infectious diseases and furnish 
a valuable aid in prognosis. 

LEUKOCYTOSIS. 

For : convenience in the study of the various forms of leu- 
kocytosis, they may be divided into two classes: (a) Physiologic 
leukocytosis, (h) Pathologic leukocytosis. 

(a) Physiologic Leukocytosis. — ^The average number of 
leukocytes, per millimeter of blood, is normally 7500. For chil- 
dren the average is slightly more. For weak and poorly nour- 
ished persons slightly less. The number of leukocytes in the 
peripheral blood of any individual vary from time to time. 
They (1) may be increased after a hearty meal, especially if it 
contains much proteid material. Physiologic leukocytosis may 
also occur (2) during pregnancy, particularly during the latter 
months of the condition; (3) in the new-born, and (4) after 
cold baths. 

In these so-called physiologic leukocytoses the increase does 
not usually exceed 30 per cent, of the normal, though in children 
it may be doubled. 

A hypo-leukocytosis is said not infrequently to precede a 
hyper-leukocytosis. 

(b) Pathologic Leukocytosis. — Many infections cause an 
increase in the number of white corpuscles in the peripheral 
circulation. Although the varieties of cells are the same as in 
health, the relative proportions are usually altered. In the more 
common forms of pathologic leukocytosis, which is seen in the 
common infections, the percentage of lymphocytes is diminished. 



10 Munch, med. Woch., September 13, 1910. 



78 CLINICAL HEMATOLOGY. 

while the polymorphonuclears are frequently increased from 
normal (65 per cent.) to 90 or 95 per cent. 

A polyw/orphonuclear leukocytosis occurs especially during 
inflammatory processes, and above all in those accompanied by 
purulent exudation. 

In certain infectious diseases, notably typhoid fever and 
uncomplicated tuberculosis, there is usually no increase in the 
number of white blood-cells. 

The origin of the extra leukocytes has not yet been defi- 
nitely determined, as we do not know whether they are derived 
from the bone-marrow, lymph-glands, or from other tissues. 

Another form of leukocytosis is characterized by a relative 
increase in the number of eosinophilic leukocytes. This condi- 
tion of the blood is notably observed in bronchial asthma, trichi- 
nosis, and infections with other animal parasites, and has lately 
been reported in Hodgkin's disease. It is of interest to note that 
in these conditions there usually exist local collections of eosino- 
philes at the seat of disease. These accumulate in the walls 
of the bronchi and in the exudate in bronchial asthma, and about 
the embryos in trichinosis. 

The number of white cells in a pathologic leukocytosis not 
infrequently reaches 20,000 to 30,000 cells per cubic millimeter, 
and has been known to reach the enormous number of 168,000 
(Grawitz). 

Leukopenia. — A diminution in the number of leukocytes in 
the peripheral blood occurs in a variety of conditions. It has 
been observed in cachexias, in intoxications, many anemias, and 
in some infectious diseases, notably in typhoid fever and in 
malaria. 

In leukopenia, as in leukocytosis, the relative proportions 
of the various varieties of white cells are usually changed. For 
example, in typhoid fever there is often a relative increase in 
the number of lymphocytes. 

THE ANEMIAS 

Anemias are conveniently classified as primary when due 
to some unknown cause, and in which the blood-changes are, 
as a rule, of both a quantitative and a qualitative type. 



THE ANEMIAS. 79 

And as secondary, when the cause is known and when the 
blood-changes are usually of a quantitative type only. 

The Secondary Anemias. — Causes: Secondary anemia is 
observed after hemorrhage, during pregnancy, during chronic 
and constitutional diseases, and in poisoning, including that large 
and vague group of conditions comprising auto-intoxication. In 
chronic digestive disorders, malignant tumors, tuberculosis, 
syphilis, malaria, and in the different forms of helminthiasis. 

The Blood-changes. — The chief blood-changes in sec- 
ondary anemia consist in a reduction of hemoglobin and a 
diminution in the number of red blood-cells. Mild forms show 
no other changes in the blood-picture. Severe forms show poi- 
kilocytosis, macrocytosis, and microcytosis. The extent to whicii 
these conditions are observed corresponds roughly to the severity 
of the anemia. 

Further, the red corpuscles often manifest a change in reac- 
tion to the ordinary staining reagents. They stain poorly, un- 
evenly, and some parts of some cells refuse entirely to take the 
stain. This condition is termed polychromatophilia. This 
change is no indication of the grade of the anemia, as it may be 
observed in the mildest forms of secondary anemia. 

The white cells in the simple anemias present nothing that 
is characteristic. 

Summary. — The essential blood-changes in the secondary, 
or simple anemias consist in a diminution in the hemoglobin per- 
centage, and in the number of red blood-cells. The red cells 
may show polychromatophilic degeneration and poikilocytosis. 

The Primary Anemias. — GtENeral Considerations: Un- 
like the secondary anemias, the blood-changes in primary ane- 
mias, besides showing any or all of the modifications observed 
in the former, present very striking and characteristic alterations 
in the white blood-cells. 

Progressive Pernicious Anemia. — In contrast to the process 
in simple anemia, in progressive pernicious anemia, blood-degen- 
eration in certain portions of the blood-making organs, notably 
in the bone-marrow, takes place in a manner different from the 
physiologic. Consequently, in the blood-formative organs and 
also in the circulation, we note cells often in great numbers that 



80 CLINICAL HE:\^LiTOLOGY. 

are never seen in the normal blood. These pathologic elements 
are present in embryonal life ; so in pernicious anemia we speak 
of the reversion of blood-formation to the embryonal type. 

The Blood-changes. — In a typical, well-defined case of 
pernicious anemia, the first glance at a well-prepared stained 
specimen of blood is sufficient to separate it immediately from 
the class of simple anemias. We find that a large number of 
erythrocytes have a diameter greater than normal (megalocytes : 
diameter 15 to 16 microns). These cells by their staining show 
a great richness in hemoglobin. Careful search always shows 
some megaloblasts, i.e., nucleated (embryonal) red corpuscles. 
Normoblasts and microcytes are also in evidence. Other changes 
usually present are poikilocytosis, polychromatophilia, and gran- 
ular degeneration. 

The red corpuscles are always notably decreased, and may 
be less than 20 per cent, of the normal. The percentage of 
hemoglobin is also diminished, but practically always to a less 
degree than the red cells. On account of this condition the 
color-index is very frequently above 1.0. 

CHLOROSIS. 

Blood-changes. — The blood when drawn flows freely from 
the puncture, and is markedly watery. Hemoglobin estimation 
shows a decided reduction in the percentage of hemoglobin with- 
out a corresponding deficiency in the number of red blood- 
cells. The color-index is therefore low, below 1.0, in contrast 
to the high color-index observed in pernicious anemia. This 
particular characteristic is not unique in chlorosis, as other forms 
of anemia may also show it. 

Morphologically we find striking changes in the erythro- 
cytes (many appear as macrocytes), which are pale and are 
without a distinctly pronounced central umbilication — the cells 
appear swollen. These particular cells have been designated 
"chlorotic" blood-corpuscles. Severe cases show poikilocytosis 
and nucleated red blood-cells. 

Polychromatophilia and granular degeneration, which are 
the genuine phenomena of degeneration, are not observed. 

The condition of the leukocytes is not uniform. The cells 



LEUKEMIA. 81 

themselves do not show any characteristic changes in this 
disease. 

The blood-plaques appear in markedly increased numbers, 
so that many groups of these cells appear in every field of the 
fresh preparation.^^ These are often very evident in the stained 
preparation, especially if a basic stain, such as methylene-blue, 
has been used. 

LEUKEMIA. 

Changes in the^ Number of Erythrocytes. — The blood from 
a marked case of leukemia is distinctly watery ; in extreme cases 
it may be a whitish-red as it emerges from the puncture; this 
is owing to the great increase in white elements. 

The microscopic examination of the fresh blood in estab- 
lished cases shows, even without counting, the enormous increase 
in the white blood-cells. The count of the white corpuscles 
shows 100,000 to 500,000, or even more, to the cubic millimeter. 

In some cases of leukemia no noteworthy change in the 
erythrocytes, either in number or appearance, occurs. As a rule 
they are decreased to about half the normal number. Besides 
the diminution in these cells the blood contains varying numbers 
of normoblasts, and more rarely megaloblasts. 

The amount of hemoglobin is diminished, but the coloring 
of the individual corpuscles need not be diminished (no altera- 
tion in the color-index). 

Changes in the Blood-plaques. — An increase in the number 
of the blood-plaques has been noted in a number of cases. 

Blood Morphology. — The diagnosis of leukemia can fre- 
quently be made without further consideration in cases which 
show an extraordinary increase in the number of white elements, 
but in doubtful cases the presence of the condition can only be 
determined by an exact examination of the morphology of the 
leukocytes. 

Besides noting the great increase in the nmnber of white 
cells, the polymorphonuclear elements will usually be found the 
most numerous. 12 in other cases the increase is chiefly among 

11 E. Grawitz : Modern Clin. Med., 1906, p. 327, D. Appleton & Co. 

12 W. von Leube : Modern Clin, Med., 1906, p. 349. 

6 



82 CLINICAL HEMATOLOGY. 

the lymphocytes. We may therefore differentiate two forms of 
leukemia. 

1. Lymphatic Leukemia. 2. Leukocytic Leukemia. 

Since the source of the lymphocytes is from the spleen and 
from the lymph-glands, and that of the leukocytes from the 
bone-marrow, the common designation of lymphatic leukemia 
(lymphemia) and myelogenous leukemia (myelemia) may be ap- 
plied to these two basic forms of leukemia. 

As, however, the bone-marrow normally produces typical 
lymphocytes, and as it has been demonstrated in rare conditions 
that an overflooding of the blood with lymphocytes may occur 
through proliferative changes in the bone-marrow without en- 
largement of the spleen or lymph-glands (myelogenous lymphe- 
mia), it seems preferable to apply the less prejudicial division 
of the leukocyte forms into "l3rmphatic leukemia" and "leuko- 
cytic leukemia'^ (W. von Leube). 

Differentiation. — 1. Lymphatic Leukemia: The blood- 
picture is conspicuous for its great preponderance of large and 
small lymphocytes, in comparison to the leukocytes. Nucleated 
red cells (normoblasts) and megaloblasts, although present in 
this form of leukemia, are by no means as abundant as in the 
leukocytic form. 

2. Leukocytic Leukemia. — This is by far the most com- 
mon form of leukemia. It may easily be differentiated from 
the preceding by the entirely different blood-picture. The in- 
crease in the white elements is usually very marked. Here, how- 
ever, the polymorphonuclear leukocytes are greatly increased 
in the microscopic blood-picture. Neutrophiles and eosinophiles 
are absolutely always increased (Ehrlich). There is also an 
increase in the mast cells, which may be twice as numerous as 
the eosinophiles. Their determination is of the greatest im- 
portance, since a marked increase in mast cells is observed only 
in this disease (von Leube). 

The phenomena which especially characterize leukocytic 
leukemia, and which result from changes in the bone-marrow 
(myelogenous leukemia), are the occurrence of neutrophilic 
and eosinophilic myelocytes. These may be present in enormous 
numbers (up to 100,000 per cubic millimeter). The first sight 



DEGENERATED RED CELLS, RING BODIES, ETC. 83 

of such a picture simulates the blood-picture of acute lymphemia 
with its large mononuclear cells. 

The myelocyte is a large mononuclear cell with an irregular 
nucleus, surrounded by a considerable amount of protoplasm, in 
which are either neutrophilic or eosinophilic granulations. Be- 
sides these immature leukocytes (myelocytes), immature forms 
of erythrocytes, also originating in the bone-marrow, are found 
in the circulating blood-stream of patients suffering from leuko- 
cytic leukemia. There are also normoblasts and occasionally 
megaloblasts. 

HODGKIN'S DISEASE (PSEUDOLEUKEMIA). 

General Lymphadenoma. — This is a variety of anemia 
characterized chiefly by progressive enlargement of the superficial 
lymph-glands. The blood-picture is that of a secondary anemia 
plus the leukocytosis occurring during fever periods, and often 
a leukopenia during the intermissions. The blood-picture in 
this disease varies with the stage. During the early part the 
hemoglobin is slightly reduced, and the red cells fall to from 
3,500,000 to 3,000,000. There may be a slight increase in the 
number of leukocytes, but this leukocytosis rarely amounts to 
more than 18,000. In the later stages there may be a great re- 
duction in both hemoglobin and red cells. 

The differential count shows an increase chiefly in the poly- 
morphonuclear element and an occasional myelocyte and some 
eosinophilia. According to Boston, in cases when the leukocyte 
count is normal, there is an increase in the percentage of 
lymphocytes. 

DEGENERATED RED CELLS, RING BODIES, ETC. 

Occasionally one observes in the red cells curious ring-like 
bodies which are very much like the hyaline malarial ring, with 
a circular refractive center. They change their shape in a 
peculiar way, much resembling the undulatory movements of 
the hyaline body. They do not increase in number or grow 
larger on standing. They are observed in a large number of 
conditions, such as measles, pernicious anemia, and severe 
secondary anemias. Herrick ^^ has observed thin, elongated, 

18 Arch, of Int. Med., vol. vi, 1910, p. 175. 



84 CLINICAL HEAIATOLOGY. 

sickle-sliaped red cells, some of them nucleated, in a severe 
anemia. 

Various other forms of degeneration of the red cells occur, 
as, for instance, the appearance of rod-like areas resembling 
bacilli, which may keep up a constant vibratory motion carrying 
them through the entire substance of the cell. This finding 
should not confuse one in making a diagnosis of the presence 
of bacteria. 

Another form of degeneration, known as Ehrlich's hemo- 
globinemic degeneration, has the appearance of a small dark 
cell lying upon a larger, paler one. These are probably areas 
of condensed protoplasm in which the hemoglobin is distinctly 
separated from the stroma. Such cells appear, occasionally, m 
certain types of malaria and are shown as corpuscles in which 
the hemoglobin is apparently condensed around the parasite. 
This degeneration is occasionally seen in nucleated red cells and 
may give the appearance of a microblast lying upon a macro- 
cyte. It is best seen in cases of pernicious anemia and may 
explain some of the "acidophilic granules^' of the red cells which 
have been described (Emerson). 

VITAL STAINING. 

Although the examination of stained specimens of the 
blood is usually made with the dried and fixed smear, excellent 
results may be obtained when fresh blood is stained without 
previous fixation. It is true that a "vitaP' staining of the blood- 
cells does not actually take place, as the dyes are decolorized by 
the reducing and oxidizing action of the living cells. However, 
a '^postvital" staining — ^that is, the staining of whole cell or 
portions of the cells, after their removal from the circulation 
and before the death of the cell results — may be accomplished in 
several ways. 

We may either add to the fresh drop of blood a very few 
crystals of the stain, as advised by Arnold, and note the stain- 
ing of certain leucocytic granules and nuclei and the reticular 
structure of many erythrocytes, or we may first dry a staining 
solution upon the slide, cover this dry stain with a drop of 
fresh blood, adjust the cover-glass, and seal this to the slide with 
paraffin or vaselin. 



VITAL STAINING. 85 

The stains which may be used for vital staining of the 
nuclei, granules, and plates are methylene-blue, toluidin-blue, 
thionin, neutral violet, Capri blue, Nile blue, brilliant cresyl 
blue, Janus green, and paraphenyl blue. Of the protoplasmic 
stains we have fuchsin, acridin red, pyronin, safranin, and 
neutral vedM 

It may be possible by this method to differentiate between 
certain forms of degeneration of the cell which are now known 
only indefinitely under the names of metachromatic and poly- 
chromatic staining (see above). 

lodophilia. — The employment of a reagent containing 
iodine is used to demonstrate the presence of glycogen, which, 
when present in the white blood-cells, particularly the polymor- 
phonuclears, is supposed to indicate the presence of a suppura- 
tive condition. Blood smears are made on slides or cover-glasses 
in the usual manner, and after drying, but without fixation, 
are mounted in a drop of iodine potassium iodide solution (see 
Appendix, page 499). 

The presence of small, brown masses in the polymorphonu- 
clears or lying free indicates a positive lodophilia. 

Significance. — These granules have been found by Hof- 
bauer in pernicious anemia, secondary anemia, and leukemia, 
but not in chlorosis or pseudoleukemia. This reaction is ob- 
served in pneumonia, but is seldom seen in tuberculosis, typhoid 
fever, and diphtheria. It is, however, not to be regarded as 
dependent upon infection, as it occasionally obtains in non- 
infectious conditions. 

Simple Test for Bile in the Blood. — A. Sunde^^ has found 
tliat it is possible to estimate the intensity of the admixture of 
bile with the blood with sufficient accuracy for all practical pur- 
poses by the length of time necessary for the color reaction to 
occur when the serum is treated with nitric acid. He used 
Scheele's modification of Gilbert's test, adding to 20 or 30 drops 
of blood-serum, in a test-tube not over 10 millimeters in diam- 
eter, a little of the reagent made by combining 300 parts nitric 
acid with 0.06 part sodium nitrite. The reagent is allowed to 
flow down the side of the tube, and a bluish ring forms at the 



^4 Webster's "Diagnostic Methods." 

15 Norsk Magazin for Laegevidenskaben, September, 1911, Ixxii, No. 9. 



86 CLINICAL HEMATOLOGY. 

junction of the two fluids. The interval before the bluish ring 
becomes evident varies from half a minute to thirty minutes 
when there is considerable bile in the blood, while with only 
small proportions the interval ranges from forty-five to sixty- 
five minutes or longer. It is possible thus to distinguish the 
pathologic from the non-pathologic cases; the shorter theinter- 
^ al, the greater the probability of some local affection in the liver 
or bile passages interfering with the normal evacuation of the 
bile. 



IV. 

SPECTROSCOPIC EXAMINATIONS. 

The spectroscope is an instrument of great valne in detect- 
ng small quantities of blood. It may also be depended upon 




Fig. 19. — Spectuoscope. 



to differentiate most accurately the various substitution products 
of hemoglobin and oxyhemoglobin; each of which produces a 
characteristic absorption band, when placed in solution and 
examined by the spectroscope (Fig. 19). 

The Spectroscope— Principle. — Light, when made to pass 
through a glass prism, is broken up into its component rays, 
giving the play of rainbow colors known as the spectrum. A 
spectroscope is an apparatus for producing and observing the 

(87) 



88 SPECTROSCOPIC exa:\iixations. 

spectrum. In brief, the apparatus consists of a base or stand, 
t^'O horizontal tubes, and a prism arranged to take the light 
coming from one and to pass it into the other (see Fig. 20). 

Light falls upon the prism through one tube known as the 
^^collimator tube." A slit at the end of this tube admits a nar- 
row ray of light which, by means of a convex lens in the other 
end of the tube, is made to fall upon the prism with its rays 
parallel. In passing through the prism the ray of light is dis- 
persed by unequal refraction, giving the spectrum. The spec- 
trum thus produced is examined by the observer through the 
other tube, which is a telescope. When the telescope is properly 
adjusted the rays entering from the prism produce a clear pic- 
ture of the spectrum. If the light used is lamplight, then the 



Fig. 20.— Direct Vision Spectroscope. 

spectrum will be continuous, the colors gradually merging one 
into the other from red to violet. If sunlight is used the spec- 
trum will be crossed by a number of narrow, dark lines known 
as "Fraunhofer lines." The position of these lines in the solar 
spectrum is fixed, and the more distinct ones are designated by 
the letters of the alphabet, A, B, C, D, E, etc. 

If, while using artificial light or the solar spectrum, a solu- 
tion of any substance which gives absorption bands is placed 
in front of the slit so that the light is obliged to traverse it, then 
the spectrum, as observed in the telescope, will show one or more 
narrow or wide, black bands, which are characteristic of the 
substance examined and which constitute its absorption spec- 
trum. The position of these bands may be located by describing 
their relation to the Fraunhof er lines. 

While the cost of the spectroscope may prohibit its use by 
the general practitioner, it is to be found in many laboratories, 
and its use in certain cases, particularly in poisoning, is abso- 
lutely essential, since it enables the student to arrive at definite 
conclusions which cannot be reached in any other way. 



HEMOGLOBIN AND ITS DERIVATIVES. §9 

HEMOGLOBIN AND ITS DERIVATIVES. 

Hemoglobin (Eeduced hemoglobin). — The absorption spec- 
trum of a solution of hemoglobin is a broad band in the yellow- 
green portion of the spectrum, extending from a little to the red 
side of D nearly to E. 

Oxyhemoglobin shows two separate bands, both between D 
and E, the one toward the green end of the spectrum being 
slightly broader and less sharply defined. 

Methemoglobin. — This substance appears in the blood and 
bloody urine of persons poisoned with potassium chlorate phen- 
acetin, other coal-tar derivatives and the nitrites, also in urine 
containing blood, after decomposition from exposure to the air. 
During the early period of decomposition of urine containing 
blood, methemoglobin shows three bands, two very similar to 
those of oxyhemoglobin and a third well defined band in the 
orange between C and D. When alkaline decomposition sets 
in the first two bands become more marked while the third band 
between G and D becomes very faint and may not be detected. 

In acid solutions, methemoglobin shows bands almost iden- 
tical with those of acid hematin (see below). For demonstra- 
tion purposes this substance may be easily produced by adding 
a little potassium ferrocyanide to a dilute aqueous solution of 
hemoglobin. 

Hematin. — Hematin varies in its spectroscopic absorption 
powers depending upon whether it is acid, neutral or alkaline. 

{a) Alkaline hematin shows general absorption of all the 
colors except a portion of the red. In dilute solutions a broad 
ill-defined band will be seen in the orange, between the lines C 
and D, extending faintly to the green side of the D line. 

Eeduced alkaline hematin (hemochromogen) shoAvs two 
lines between I) and E, but extending more toward the blue 
side of the E line. 

(&) Acid Hematin. — This may be produced by treating the 
blood or dried bloody debris with a mixture of alcohol and 
ether acidified with glacial acetic acid. The cloudy emulsion 
produced may be cleared by increasing the alcohol or ether pro- 
portion. The absorption spectrum shows four distinct bands, 
one well defined between C and D lines toward the C end of the 



90 SPECTROSCOPIC EXAINIINATIONS. 

spectrum^ three between the D and E lines, of these the one 
nearest the D line is narrower than the other two. 

Chlorophyl. — ^Vegetable coloring matter which may be 
present in vomitus or in feces, gives the same absorption spec- 
trum. This substance may be differentiated from acid hematin 
by adding a few drops of 10 per cent, sodium hydrate solution 
and some ammonium sulphite to this solution. This changes the 
spectrum of a blood-containing solution to that of reduced 
hematin, while the chlorophyl spectrum absorption remains 
unchanged in spite of the addition of a reducing agent. 

CO-hemoglobin. — The absorption spectrum of this deriva- 
tive closely resembles that of oxyhemoglobin but can be fairly 
well differentiated by the fact that the bands are moved slightly 
nearer the violet end of the spectrum, and do not become fused 
into a single band when the solution is treated with ammonium 
sulphite or other reducing agent. Amounts less than 20 per 
cent, of CO-hemoglobin in the suspected blood cannot be de- 
tected by the spectroscope.. 

Cyanhemoglobin. — In testing for blood in aJl stains when 
the suspected material has been rendered soluble by potassium 
cyanide, the solution obtained gives an ill defined broad absorp- 
tion band in the yellow-green portion of the spectrum between 
the lines D and E. Methemoglobin when acted upon by a 
cyanide gives a broad band of absorption quite similar to that of 
cyanhemoglobin. 

Examination" for Blood in Stool. — A portion of feces is 
extracted with ether to remove the fat and then separated from 
the ethereal extract in the usual way. The remaining fat-free 
feces is rubbed up with water in a small mortar and treated 
with 2 volumes of ether and an equal quantity of acetic acid. 
The ether takes up the hematin which can, be detected in solu- 
tion by the spectroscope, v/hen it gives the characteristic 4 
bands (see Plate III). To differentiate the spectrum of 
chlorophyl from that of hematin, add a 10 per cent, solution of 
potassium hydrate and ammonium sulphite and note the 
change indicated above. 

Urobilin. — This substance is soluble in ethyl alcohol, 
chloroform and amylic alcohol and but slightly soluble in 
ether, acetic ether and in water. 



PLATE III. 



Oxyhsemo 
globin 




Cyanmet- 

hsemoglo' 

bin 



Photomet- 
haemoglo- 
bin 



ABSOUI'TION Sl'KCTKA OF HEMOGLOBIN AND ITS DERIVATIVES. 



ton 



(Prom Wood's Chemical and Microscopical Diagnosis. Courtesy of D. Apple- 
& Co., Publishers.) 



HEMOGLOBIN AND 1T8 DERIVATIVES. 91 

Technic. — Add a few drops of HCl to 25 cubic centimeters 
of urine and shake out with 10 cubic centimeters of amylic alco- 
hol with the aid of a separatory funnel (Fig. 54). This acid 
solution in the presence of urobilin will show an al)sorption 
band keeping somewhat to the right of E and terminating on 
the right beyond the F line. 

TJroroseinogen (chemicall}^, indol-acetic acid) is converted 
by oxidation into the pigment urorosein which is soluble m 
ethyl and amylic alcohol but insoluble in ether, chloroform and 
benzol. 

To detect, extract 20 cubic centimeters of urine with 5 cubic 
centimeters of amylic alcohol, when spectroscopic examination 
will show a sharp but narrow absorption band between"!) and E. 

Stokes's solution, reducing agent to use in place of ammonium 
sulpliid sohition. 

Ferrous sulphate 2 Gm. 

Tartaric acid 3 Gm. 

Water 100 c.c. 

To use, place portion in a test tube and add ammonium hydrate 
until the precipitate which first formed has entirely aissolved. This is 
then ammonium ferrotartrate, which is the reducing agent. 

Alkaline hematin shows one absorption band between C and 
D, which extends through the line D, toward the line E. The 
additions of Stokes's solution changes the alkaline hematin to 
liemochromogen which shows two characteristic absorption 
bands, one sharp and dark between D and E and a second 
fainter and broader covering the line E. 

Hematoporphyrin. — The treatment of hematin with con- 
centrated H2SO4, in the presence of air, splits off' the iron 
leaving the pigment hematoporphyrin. This is isomeric with 
bilirubin. It is insoluble in water but dissolves in alcohol, alka- 
lies and in acid. 

Hematoporphyrin in acid solution shows two absorption 
bands, one faint and narrow between C and D but closer to D : 
another darker, more definite and broader midway between D 
and E. In dilute aika'ine solution there will be seen four ab- 
sorption bands (a) between C and D^ (h) broader band covering 



93 



SPECTROSCOPIC EXAMINATIONS. 



D extending well into the space between B and E, (c) between D 
and E close to E and {d) a broad and dark band between E 
and F. 

IVES REPLICA DIFFRACTION GRATING. 

Gordon J. Saxon^ describes a simple and useful device 
which serves practically the same purpose as the more expensive 
and complicated spectroscope. The examination of dilute solu- 
tions is successful because it is possible to prolong the length of 
the fluid column, thereb}- gaining the same advantage in 
analysis as would come from concentration of the fluid. The 
column of fluid can, when necessan*, be lengthened to 1 meter. 



K 



M 



N L F 



^f^ 



Fig. 21.— Set Up of Ives Grating, 



under which conditions Saxon reports the demonstration of 
bands of absorption from oxyhemoglobin in a solution contain- 
ing 1 drop of blood in 2 liters of water. 

Preparation of Apparatus. — As indicated in Fig. 21, G is 
the Ives' grating (obtainable from any instrument supply house, 
cost $12 in 1914) held in an}^ suitable frame; 8 is any opaque 
substance (thin sheet metal is best) having a longitudinal slit 
in it for the transmission of the light rays; CK is a light 
screen of carboard or other light but opaque substance, placed 
so as to shield the eyes of the observer from any extraneous 
light rays, having a perforation large enough to permit the 
light rays to pass through the slit in 8 without interference. 
MN is the trough which may be of any convenient length up to 
1 meter ; this also should be supported in a suitable clamp-frame 
or stand, which will hold it firmly in the optical axis of the 



1 Saxon : Jour. Biol. Chem.. 1914, vol. xvii, 2, p. 103-106. 



IVES REPLICA DIFFRACTION GRATING. 



93 



apparatus between G and F, as shown by the dotted line ; 7^ is a 
double convex collecting lens of sufficient diameter to cover the 
end of the specimen tube, having a focal length of from 15 to 
20 centimeters, and so arranged before the source of light F, 
that the center of F is the focal point of the lens L ; this set-up 
delivers parallel rays of light through the apparatus to the 
grating at G. The slit in the diagram at S is 0.5 millimeter 
and should be clamped so as to permit up and down adjustment. 




FIG. 22.— MEASUREMENT OF WAVE LENGTH. 



The distance from the outer end of the trough to the diaphragm 
should^ be as short as possible, while the distance from the 
diaphragm to the grating G is arbitrary, although if it is desired 
to use the apparatus for measurements the distance SG, should 
be at leasti 1 meter. 

Employment of Set-up. — Looking straight through the 
grating which is transparent, the slit S can be seen, on either 
side of this direct image will appear spectra, the whole color 
dispersion being visible at the same time. The absorption spec- 
trum does not consist of one or more sharp areas of color miss- 
ing from the continuous arrangement of colors, because not only 
is a color of one wave length absorbed, but also some adjacent 



94 SPECTROSCOPIC EXAMINATIONS. 

to this area on both sides. The width of the bands will vary 
with the concentration of the fluid and the length of the 
column through which the light passes. 

Comparison of the absorption spectrum can easily be made 
with the direct or unabsorbed spectrum of the same source of 
light. This is accomplished by permitting some of the light 
rays from F through the lens L to pass above the trough MN, 
and so reach the diaphragm 8 without passing through the fluid. 
The diaphragm is raised sufficiently to allow the passage of 
these direct rays, so that they fall coincidently upon the grating. 
The result will be two parallel spectra visible to the observer at 
the same time.. This plan renders the absorption spectrum 
most striking. 

If it is desired to measure the wave lengths of the absorp- 
tion bands a meter-stick may be placed along S, as in Fig. 22. 
In looking at the spectrum the position of the band can be 
noted on the meter scale and the distance a, determined. It is 
well to obtain this position on both sides of 8, and to employ 
the average value thus obtained. The wave-lengih of the par- 
ticular band located at a is calculated by the formula: 

/ = -A^ or / = 



l/a2 + b2 

In which I =^ wave length, a = distance, along the meter stick, 
from the slit 8 to where the band is seen, d = distance be- 
tween lines on the diffraction grating. 

In purchasing a diffraction grating the number of lines 
ruled per centimeter or per inch, should be obtained from the 
maker. With age this distance may change, the grating should 
therefore be tested occasionally by means of light of known 
wave-length, e.g., sodium flame. 



V. 

NEWER METHODS OF BLOOD 
EXAMINATION. 

Medical progress has forced the author to digress from 
the plan laid down in the preparation of the earlier editions of 
this book. The intention of this work as expressed in the earlier 
editions was to present a limited number' of alternative tests in 
a series of methods having a minimum of technical diihculties, 
feeling that the methods presented would be sufficient to permit 
the general practitioner to efficiently and thoroughly study his 
cases with the least expenditure of time and a minimum knowl- 
edge of laboratory technic. 

During the past few years the progress of scientific clinical 
medicinq has been so rapid, chiefly through the development of 
many new and clinically valuable methods, that the general prac- 
titioner has been forced more and more to depend upon the 
laboratory for information andl guidance. Most of these highly 
technical procedures involve the study of the chemistry of the 
blood on a basis of alterations in blood composition as modified 
by changes in body metabolism. 

At the risk of including technic, which may by some be 
(Considered beyond the scope of the physician, the author has 
grouped in this chapter, for want of a better classification, 
methods for the determination of coagulation of the blood, vis- 
cosity of the blood, blood urea, blood sugar, cholesterol content 
of the blood, non-protein nitrogen of the blood and the alkali 
reserve of the blood plasma. Some of these methods require 
considerable skill and practice. Others require rather extensive 
apparatus, but it is hoped that the details and directions herein 
found have been made sufficiently plain and concise to reduce 
technical difficulties to a minimum. 

A. COAGULATION TIME OF THE BLOOD. 

Much interest has been manifested recently in the theo- 
retical and clinical value of the study of the coagulation time 

(95) 



96 NEWER METHODS OF BLOOD EXAMINATION. 

of the blood. A review of literature shows a large number of 
methods, together with their modifications, which have been 
devised in an effort to render this property of the blood of 
practical clinical value. This review shows that the results ob- 
tained by competent observers using the latest methods and 
apparatus have sufficient similarity to form an accurate clinical 
basis upon which confidence may be placed. The apparatus 
devised for determining the clotting time are of three distinct 
types : 1. The capillary tube which forms the basis of the Wright 
method. 2. The apparatus in which corpuscular motion is 
observed to cease under a current of air, as in the methods of 
Eussell and Brodie and of Boggs. 3. Those dependent upon the 
contour of the" drop of blood. 

This discussion will be limited to a selected few of the 
many methods employed, because a multiplicity of methods 
tends to confuse the worker, and because the methods following 
have been found by the author to fulfill all clinical requirements. 

It is essential in the study of the clotting time of the blood 
to bear in mind the various factors entering into and modifying 
the results of these observations. According to Dorrance and 
others, these may roughly be classed as extrinsic and intrinsic. 
The extrinsic factors are as follows: — 

(a) Foreign body, as dust or dirt. 

(&) Area of surface contact. 

(c) The air evaporation (humidity). 

(d) Temperature. 

(e) Size and shape of drop. 
(/) Mechanical disturbances. 

(g) Determination of end point (personal oquatioii). 
The intrinsic factors are as follows : — 

1. Those affecting the blood. 
(a) Viscosity. 

(h) Blood-pressure. 

(c) Leukocytes. 

(d) Specific gravity and anemia. 

(e) Chemical composition of blood. 

2. Physiologic conditions of the body. 

(a) Sex and age. 

(b) Menstruation period. 



COAGULATION TIME OF THE BLOOD. 97 

(c) Time of day. 

(d) Diet. 

To this may be added the region from which the blood is 
drawn, the shape, size, and depth of the puncture, contact with 
vital tissues, and accidental contact with previously shed blood 
or an old clot. 

THE SPECIMEN. 

This should be taken midway between meals, and not 
after the ingestion of drugs, which may influence coagulation 
time. The tips of the finger or the lobe of the ear should be 
thoroughly cleansed and dried, and a sufficiently large puncture 
made with a flat-bladed instrument (Daland lancet) to insure 
free and sufficient flow. 

METHODS OF DETERMINATION. 

Milan's Method. — Place a drop of blood upon a large glass 
slide or in the center of a watch-crystal. After a minute or two 
gently tilt the glass from side to side. This is continued at 
short intervals until the drop of blood, which at first assumed a 
pear shape when the glass was tilted, is seen to assume the form 
of a blunt and symmetrical cone. This change in the character 
of the drop indicates the completion of coagulation. The aver- 
age time of coagulation of normal blood by this method is about 
five minutes. 

This method is at best imperfect and uncertain, since none 
of the modifying factors are controlled. 

Method of Eussell and Brodie. — This method requires a 
microscope. The coagulometer consists of a small, moist cham- 
ber with a glass bottom, which can be placed upon the 
stage of the microscope. This is fitted with a truncated cone of 
glass, which projects downward into the moist chamber. The 
end surface of this cone is of definite size (about 5 millimeters 
in diameter), and on it is placed the drop of blood, care being 
taken to see that the drop of blood only just covers the surface; 
hence is always of the same size. The cone is then quickly fitted 
into the moist chamber to prevent alterations due to drying, 
temperature, etc. Through the side of the moist chamber pro- 
jects a fine tube, through which, by means of a hand-bulb, a 

7 



98 NEWER IMETHODS OF BLOOD EXAMINATION. 

gentle stream of air can be projected against the blood. The 
preparation of the slide being completed, the drop of blood is 
observed with the low power of the microscope. 

The blowing should be done at as long intervals as possible, 
and also very gently. The corpuscles will first be seen to move 
freely as individuals; later, as coagulation begins, the corpuscles 
will no longer move freely in the drop, but the drop will begin 
to change shape en masse. Finall}^, there will occur only elastic 
motion, and the part of the drop displaced by the current of air 
will spring back to its original position as soon as the current 
of air ceases. This is to be taken as the terminal point, as now 
only can the clot be demonstrated by quickly removing the cone 
and touching the drop to a piece of dry filter paper. 




Fig. 23.— Boggs's Coagulometer. (A. H. T. Co.) 

Eecently this instrument has been improved upon by Boggs, 
who has substituted a metal tube and an improved cone (see 
Fig. 23). 

With the improved instrument Emerson ("Clinical Diag- 
nosis") reports normal variations in the coagulations between 
three and eight minutes, with an average of five minutes eight 
seconds. 

Method of Dorrance.^ — The coagulometer of Dorrance is 
composed of an 8-ounce thermos bottle without the silver lining, 
an aluminum stand, and two rubber corks which fit snugly into 
the bottle. One cork has a hole in it for a thermometer which 
registers up to 140° F. The other cork has four openings, each 
of which is lined with a brass flange. Into each of these open- 
ings a glass rod fits. These rods are 4% inches long, with a 
diameter of 8 millimeters. One end is cone-shaped, with a flat 
tip 4 millimeters in diameter. The other end is slightly bul- 
bous, to prevent the rod from slipping through the flange. 

1 Tte description of this instrument is taken from the deviser's article ap- 
pearing in the American Journal of Medical Science, October, 1913, No. 4, p. 562. 



COAGULATION TIME OF THE BLOOD. 99 

Method of Application. — The thermos bottle is filled with 
water at 98° F. to within one inch of the top. It is then covered 
with the cork containing the thermometer to note any change 
in the temperature of the water. The rods within the second 




Fig. 24.— Dorrance's Coagulometer. 



cork are first scrubbed with soap and water and then cleansed 
with alcohol and finally with ether. This cork is then sub- 
stituted for the one containing the thermometer, so that the 
rods will become heated to the temperature of the water. This 



100 NEWER METHODS OF BLOOD EXAMINATION. 

is done while the finger of the patient is being prepared. The 
rods during the time that they are in the bottle heating up to the 
temperature of the water will have collected considerable mois- 
ture, and it is therefore necessary to wipe them dry before col- 
lecting the blood, else the moisture will dilute the drop of blood. 
The finger is given a good stick, so that it bleeds without pres- 
sure. The first drop of blood is wiped off. As soon as another 
drop begins to appear the cork containing the glass rods is 
removed from the bottle and the rods are wiped dry. Then 
with care each one of the rods is placed in contact with the 
drop of blood. If done carefully the same sized drop will be 
taken up by each rod. From the time of the appearance of the 
drop until the blood is collected and the cork replaced in the 
bottle should not be more than ten seconds. 

The end point with this instrument is determined in three 
ways: 1. In explaining the end results we shall number the 
glass rods in the order in which the blood was collected upon 
them. At the end of two and one-half minutes rod No. 1 should 
be pushed down into the water. It will be noted that all the 
blood falls off the end of the glass rod and breaks up into a fine 
cloud. Eod No. 2 is introduced into the water at the end of 
three and one-half minutes. There will still be considerable 
falling off of the blood, but when it breaks up the particles will 
be coarser and there will remain a small amount on the end of 
the rod. Eod No. 3 is introduced into the water at the end of 
four minutes. There will still be noted some dropping off of 
the blood, and there will be found a larger amount of blood ad- 
herent to the end of the rod. Eod No. 4 is introduced into the 
water at the end of four and one-half or five minutes, depending 
upon the action of the former rods. There will be a slight fall- 
ing off, but the greater part will be found to be adherent to the 
end of the rod if coagulation has taken place. The fact that we 
start at two and one-half minutes from the time of the appear- 
ance of the drop is only an arbitrary factor. The test can begin 
at any time and the rods be introduced at any time that the 
operator desires. 

2. The cork containing the four rods is now removed from 
the bottle and held up to the light, with the end on which the 
blood is adherent nearest to the eye. On looking through the 



COAGULATION TIME OF THE BLOOD. IQl 

rods the following results will be found : Eod No. 1 will be clear. 
On Eod Ko. 2 will be observed a slight reddish tinge. On Eod 
No. 3 this reddish tinge will be somewhat deeper in color and the 
end of Eod No. 4 will be almost covered with a red clot. 

3. The end of each rod is blotted with filter paper or 
blotting paper. The blot made by , Eod No. 1 will be prac- 
tically clear. Eod No. 2 will show a slight red color, Eod 
No. 3 will show a more distinct red, and Eod No. 4 will show 
a clot. If the last rod does not show the blood to have coagu- 
lated, the test should be repeated, with the first rod introduced 
at four minutes and the others at either one-half minute or 
one-minute intervals. 

It is a well-established fact that the first drop does not 
coagulate as rapidly as the later ones, and, as we want to know 
the shortest time, we take about the fourth or fifth drop. Blood 
that is taken from the veins and which does not come in con- 
tact with the tissue juices does not give the true clinical coagula- 
tion time. The tissue juices are one of the most important fac- 
tors in the causation of coagulation. 

Pathology. — The pathologic significance of coagulation time 
clinically is as yet indeterminate. In typhoid fever it is vari- 
able, being more rapid especially in cases with thrombosis. It 
is rapid in leukemia, diabetes, endocarditis. It is delayed in 
hemophilia, purpura hsemorrhagica, lymphatic leukemia, and 
splenic anemia. In diseases of the liver and gall-bladder the 
decreased coagulation seems to vary directly with the depth and 
intensity of the jaundice. Of the acute infections, lobar pneu- 
monia and septicemia show the most consistent and longest 
delay of clotting. 

On the other hand, T. Addis,2 found in examining a large 
and varied group of diseases that in 70 per cent, the coagulation 
time was not disturbed. Moderate loss of blood has no effect 
on coagulation, but after large hemorrhage the coagulation time 
is accelerated. 

Eudolph^ says that of the effects of drugs upon the coagu- 
lation he found calcium lactate did not appear to have any 
effect, and that citric acid appeared to have a slight retarding 
influence. 



2 Edinburgh Med. Jour., July, 1910. 

3 New York Medical Journal, August 13, 1910. 



102 NEWER METHODS OF BLOOD EXAMINATION. 

In the study of coagulation it must be remembered that the 
personal equation and large variety of apparatus affect the de- 
termination of the end point, and that it is difficult to make an 
exact comparison of the results of different workers. It would 
seem advisable that some uniform procedure should be devised 
and adopted, thereby minimizing the effect of the factors of 
error, and yet which would be sufficiently practical for all clin- 
ical purposes. 

The work of Eobertson and lUman, while having been done 
some years ago, still remains unchanged. They employed the 
cumbersome Wright apparatus and obtained the following re- 
sults, which are based upon many observations of a large num- 
ber of patients: — 

Specific meningitis 3,10 minutes. 

Coal-gas poisoning 4.05 " 

Gastric ulcer 4.10 " 

Salpingitis 5.15 " 

Alcoholic gastritis 3.10 " 

Apoplexy 3.05 " 

Carcinoma 4.35 " 

Jaundice 6.30 to 8.30 " 

Rheumatism 3.00 to 4.00 " 

Pneumonia 2.47 " 

Nephritis 2.25 

Diabetes 2.55 " 

Exophthalmic goiter 2.45 " 

B. VISCOSITY. 

The great activity in the study of blood-pressure during the 
past decade has served to emphasize the importance of blood 
composition in relation to its passage through the vessels. 
In the section on blood-pressure will be found discussed the fac- 
tors determining both normal and pathologic blood-pressure, and 
among them we find two which are chiefly concerned in determin- 
ing the peripheral resistance to the circulation of the blood. 
These are: 1. The ever-changing caliber of the arterioles and 
capillaries. 2. The viscosity of the blood. 

According to Huertel, the work done by the heart of a 
dog is four times greater than it would be if the vessel were 
filled with water. This difference is due to the viscosity of the 



VISCOSITY. 103 

blood, which is a physical property depending on the freedom 
of motion between the particles which constitute the blood and 
which, according to different investigators, in the normal human 
is 3.5 to 5.5 times less than that of water. We are not par- 
ticularly concerned with the importance of the individual 
factors which combine to produce the normal viscosity of the 
blood. Some of this data is still in the speculative stage, and 
has not yet been sufficiently studied to be of clinical importance. 

A rather interesting theory is that of Adams, who maintains 
that the viscosity is dependent primarily upon the amount of gas 
which the blood contains, saturation with carbon dioxide pro- 
ducing a maximum of viscosity. If the carbon dioxide be 
replaced by oxygen then the internal friction is reduced to a 
minimum. 

There is probably a relation between the viscosity and the 
fibrin in the blood, as it has been noted that viscosity and coagu- 
lability increase and diminish pari passu within certain limits. 
We do not believe that the viscosity of the blood is altogether 
dependent upon the specific gravity because of the fact that high 
viscosity may coexist in fevers with a low specific gravity. For 
definite physical reasons, the amount of protein and salt con- 
tained in the plasma greatly influences the viscosity, and it is 
probable that among drugs the iodides are the most important, 
as they appear to have a powerful effect in diminishing 
viscosity. 

Factors Affecting Viscosity. — ^The viscosity of a normal 
individual varies according to age and sex. But there exists 
a difference of opinion among investigators as to the average 
viscosity for different ages of healthy individuals. In infants 
it probably varies between 3.2 and 3.6, reaching 3.9 to 4.2 at 
about 10 years, being between 4.2 and 4.5 between 12 and 20. 
The average of all investigators places it between 4.5 and 5.1. 
Blood viscosity is a relatively constant factor, showing that vari- 
ations above 4.4 and 5.3 in men and 3.9 to 4.9 in women must 
be considered pathologic (Sachs). It is believed that viscosity 
is slightly lowered in males after the fiftieth year. Blunschy 
states that muscular exertion, when severe and prolonged, re- 
duces viscosity, but that when mild and comparatively brief, even 
when causing perspiration, it increases it. 



104 NEWER METHODS OF BLOOD EXAMINATION. 

It is generally believed that meat-eaters have a relatively 
higher viscosity than vegetarians, although the difference is but 
slight. The ingestion of large quantities of water and food ap- 
pears to temporarily influence viscosity. 

Principle of Determining Viscosity. — The fundamental 
principle in any apparatus for determining the viscosity of the 
blood is that of forcing blood through a given length of 
capillary tubing under a definite pressure and temperature and 
comparing the time consumed with that required to force dis- 
tilled water through the same tube under like conditions. For 
this purpose McCaskey has devised a very simple and practical 
apparatus, which, while devoid of absolute scientific accuracy, 
fulfills the requirements of clinical medicine. 

Method of McCaskey. — ^The instrument consists of a capil- 
lary pipette made from a piece of glass tubing with a lumen of 5 
millimeters. One end is tapered so that the rubber bulb can be 
easily fitted on it. The other end is drawn out into a fine 
capillary tube, the exact size of which must be determined by 
experience. This capillary tube is substantially the same as the 
capillary pipette used in opsonic work. A convenient length 
for the capillary portion of the tube is about 6 inches, and it 
should be made so that distilled water will pass through it 
under the negative pressure of the bulb in about five or six 
seconds. A fine inkmark is made at 5 inches from the tip just 
before the lumen of the capillary tube begins to enlarge, and 
each experiment terminates at the moment when the moving 
column of blood reaches this mark. It is first necessary to 
"standardize^^ the tube with distilled water, which constitutes 
the unit of comparison. The rubber bulb is taken between the 
thumb and index finger and compressed until its inner surfaces 
are in contact. It is important that this technic should be 
accurately followed. If this be done there will not be any sub- 
stantial variations in the negative pressure of the same bulb in 
different experiments. If, however, the bulb is compressed 
flatly, say, between the palmar surfaces of the thumb and two 
fingers, the negative pressure will be increased from 40 to 50 
per cent, and the results entirely vitiated. The negative pres- 
sure will vary with different bulbs. The bulb thus compressed 
is fitted over the capillary pipette at the end of the tube and the 



VISCOSITY. 105 

pressure released. Air flows into the capillary tube under a 
constant negative pressure. Holding a stop-watch in the other 
hand, the end of the pipette is immersed in distilled water con- 
tained in a watch-crystal and the stop-watch started at the same 
instant. The watch is stopped when the column of water reaches 
the mark. This is repeated three or four times, and, if there be 
any variation in the time, the mean of the several observations 
is taken and a label is attached to the tube showing the result 
and also the temperature of the room. The latter is important 
because the viscosity falls about 2 per cent, with each degree 
centigrade of rise of temperature; and if the observation on 
the blood is made at a temperature substantially different from 
that in which it was standardized with distilled water, cor- 
responding corrections must be made. 

The clinical observation of the blood viscosity is made as 
follows: One arm of the V-shaped tube (made of glass tubing 
with a lumen of 1.5 millimeters) is partially filled with blood by 
holding it against the under surface of a drop of blood exuding 
from the side of the tip of the finger or from the lobe of an ear. 
Five or 6 drops are required. This tube is then held by an 
assistant, or in a suitable clamp attached to the arm in which the 
blood has been collected, leaving the clear arm of the tube free 
for observation. The bulb and capillary tube are arranged pre- 
cisely as heretofore described for the experiment with distilled 
water, and the end of the capillary tube is passed down the clean 
arm of the V-shaped tube until it comes close to the surface of 
the specimen of blood. Then, while holding the stop-watch in 
the other hand, it is quickly plunged into the blood and the 
stop-watch is started at the same instant, the watch being 
stopped as before when the moving column of blood reaches the 
inkmark. It is then simply a matter of dividing the time re- 
quired by the blood to pass through the capillary tube by the 
time required by the distilled water to detemiine the viscosity. 
If, for example, with a particular pipette, it would necessitate 
twenty-five seconds for the blood to reach the mark, while dis- 
tilled water requires five, then the viscosity would be five. This 
observation can be made in three minutes and is sufficiently 
accurate to serve the purpose of clinical medicine. Sometimes 
the blood coagulates so rapidly that only a single observation 



106 NEWER METHODS OF BLOOD EXAMINATION. 

can be made. This can be obviated by placing a crystal of 
hirudin into the V-shaped tube, as recommended by Bence and 
Determann. The blood will then remain fluid for twenty or 
thirty minutes. The greatest objection to the use of hirudin is 
that it is very hygroscopic and must be kept hermetically sealed, 
or in a chamber with a desiccator. Its use, however, does not 
change the viscosity in the least or even modify rouleau forma- 
tion, so that it is available for other purposes in which slow 
coagulation is desirable as well as the determination of viscosity. 
The viscosity tube can be cleaned in the usual way with water, 
alcohol, and ether. If the tube is spoiled it can be easily and 
cheaply replaced. 

Method of Benning and Watson. — A second method, which 
is very simple, the apparatus for which may be obtained on the 
market, is as follows :^ It consists of a J-shaped capillary tube, 
the long arm of which measures 6 and the short arm 2 centi- 
meters in length. The upper end of the former is molded so 
as to form a funnel-shaped receiver, and in the course of the 
short arm the tube is blown to form an elliptical bulb. The 
points where the capillary bore meets and leaves the bulb are 
marked on the glass. To use the instrument the lobe of the ear 
is pricked (aseptic precautions being observed), and the instru- 
ment, which has been warmed to the body temperature, is held 
under the exuding drop of blood, so that it flows into the re- 
ceiver. The flow down the tube is carefully watched until it 
reaches the mark indicating its entrance into the bulb. The time 
is immediately noted on a stop-watch, and again when the bulb 
is filled, when it is read off to the fraction of a second. The 
instrument is calibrated for water, the viscosity of which is 
known, and the time value is marked on the back of the tube. 

Factors Affecting Viscosity. — Carbon dioxide increases vis- 
cosity. For this reason asphyxia more promptly affects vis- 
cosity than any other known factor, whether this be general, 
or a local condition due to blood stasis. 

Experiments in asphyxia have shown viscosity to increase 
from 4.63 to 8.83 (Determann). A viscosity factor of 12 was 
obtained by Trumpp in a newborn infant in blue asphyxia. 



4 A. Denning and J. H. Watson: Lancet, July 14, 1906; and Liverpool 
Med. Chir. Jour., July, 1906, p. 46. 



NEWER BLOOD CHEMISTRY. 107 

Slight exertion in persons with cardiovascular disease markedly 
increases viscosity. A number of observers state that an increase 
in viscosity is an early sign of cardiac insufficiency and suggest 
the importance of this observation as an aid in early diagnosis. 
In this connection it has been stated that patients with increased 
viscosity will, as a rule, die, while those with a low viscosity 
can be given a better prognosis. 

Venesection lowers viscosity, and its value as a therapeutic 
measure is probably in a large measure dependent upon this 
relief afforded the heart. Intravenous administration of salt 
solution reduces viscosity; so also does hemorrhage. The effect 
of the former is of short duration, while that of the latter quite 
lasting. Viscosity is high in pneumonia and in emphysema and 
low in anemia and impoverished blood conditions. 

The relation of viscosity to renal diseases is particularly 
important. It is now believed that in chronic interstitial 
nephritis the viscosity is low, even in the presence of a high 
blood-pressure. From the clinical standpoint a rather impor- 
tant observation is that of Trumpp, who observed a rather high 
viscosity in children with alimentary intoxication. From the 
surgical standpoint, the viscosity is high in clean abdominal 
operations during the first two or three days, and gradually 
falls to and below the normal, returning to normal at the end of 
a week. The same change is apparently present in septic cases, 
where the inflammation is relieved, but remains high if the in- 
fection persists. In typhoid fever the viscosity is usually low, 
while in meningitis it is generally increased. The effect of 
drugs is most marked. Alcohol greatly increases the viscosity. 
Caffeine also increases the viscosity, while camphor reduces this 
factor. Chloroform and ether do not seem to have any effect 
on viscosity. 

C. NEWER BLOOD CHEMISTRY. 

DETERMINATION OF BLOOD UREA. 

Method of Marshall — Principle. — Marshall states^ that 
the soy-bean extract described (page 108) in determining the 
percentage of urea in the urine is not directly applicable to the 
quantitative determination of the urea in the blood serum, be- 



5 Marshall : Jour. Biol. Chem., xv, 3, 487, 191c 



108 



NEWER METHODS OF BLOOD EXAMINATION. 



cause of the large quantity of proteins here present in comparison 
with the small amount of urea. This difficulty is overcome by 
first, converting the urea into ammonium carbonate by the use 
of the enzyme, and subsequently removing the ammonia from 
the solution by means of a current of air, and finally deter- 
mining the percentage of urea by titrating the amount of 
ammonia formed. 

Procedure. — The blood is drawn in any convenient man- 
ner and allowed to stand on ice until coagulation is complete. 
Two equal portions of serum are measured into ordinary test 
tubes, 1 cubic centimeter of the soy-bean extract or one or two 



Vnttp 




Fig. 25. 



25-milligram urease tablets added to one tube and about 0.5 to 
1.0 cubic centimeter of toluene to each. If sufficient serum is 
available 10.0-cubic centimeter portions should be used, how- 
ever, , perfectly satisfactory results can be obtained by using 5.0- 
cubic centimeter or even 3.0-cubic centimeter portions of serum. 
The tubes are tightly stoppered and allowed to stand at room 
temperature over night until conversion of the urea into am- 
monium carbonate is complete. The contents of the tube con- 
taining the serum and enzyme solution is transferred to cylinder 
A, and washed in with not more than 5 cubic centimeters of 
distilled water (Fig. 25) . 

Two grams of sodium chloride, an equal volume of water 
and a layer of kerosene -oil are added to the cylinder. The 
contents of the other tube is transferred to cylinder B, and 



NEWER BLOOD CHEMISTRY. 



109 



treated in exactly the same manner. Twenty-five cubic 
centimeters of N/50 HCl and about 25 cubic centimeters 
of distilled water are placed in each of the 200-cubic centim- 
eter Erlenmeyer flasks, which are for the purpose of absorbing 
the ammonia. When completed the different parts of the 
apparatus will appear as shown in the cut. One-half a gram 
of sodium carbonate is added to each cylinder. The last Erlen- 
meyer flask is connected to a suit- 
able suction pump, and a current of flA.vA.T.co — 
air is allowed to flow through the 
apparatus until all the ammonia 
has been removed from the cylinder. With a 
good pump this takes about 1 hour. The excess 
of acid in the absorption flask is titrated with 
]^/50 sodium hydroxide, using as an indicator 
sodium alizarin sulphonate. The amount of acid 
neutralized in the flask attached to cylinder B 
corresponds, of course, to the ammonia present in 
the serum, while the amount used in the other 
tw^o flasks represents! the urea and the ammonia. 
The difference, therefore, is the amount of urea- 
ammonia in the blood in terms of N/50 HCl, 
which, when multiplied by 0.0006 gives the urea 
in grams present in the original amount of serum 
taken for the determination, from which the per- 
centage of grams per liter are readily determined. 

Detailed Description for Setting-up Ap- 
paratus. — The tubes c and c' are ordinary cal- 
cium chloride tubes packed loosely with cotton. 
These in conjunction with; the bulbs prevent any 
splashing and mechanical transmission of the 
alkali into tlie absorption flasks. The bulbs are not absolutely 
necessary but aid in keeping the cotton filters dry. 

For the better absorption of the ammonia, the long tubes 
in the Erlenmeyer flasks are closed at the lower end and pierced 
with G or 7 small holes (Fig. 26). Because of the necessity of 
fully absorbing the ammonia, 2 Erlenmeyer flasks should be used 
in' combination with the cylinder in which the urea-ammonia is 




FIG. 26. 

FOLIN 

Ammonia 

Absorption 

Tube. 



110 



XEWER METHODS OF BLOOD EXA^IIXATIOX, 



liberated, while on account of the small amount of ammonia in 
the serum cylinder one flask is sufficient. 

The bottle D contains dilute H2SO4 to free the air of any 
traces of ammonia before passing it through the apparatus. 

Xo correction is necessary for the ammonia derived from 
the 1.0 cubic centimeter of sov-bean extract used, as the amount 



To Suction 




-Wa5h Bottle 



Fig. 27.— VAX Slyke and Cullex Urea apparatus. 



obtained from the source is negligible. Foaming is prevented 
by the use of caprvlic alcohol, toluene or kerosene in the cylin- 
ders and toluene in the flasks. If foaming is not prevented a 
loss of a portion of the contents of the flasks and cylinders will 
occur. 

Van Slyke and Cullen^ modification of ]\Iarshall"s method. 

Peoceduee. — Prepare a 100.0-cubic centimeter test-tube by 
adding 1.0 cubic centimeter of a 3 per cent, solution of potas- 
sium citrate to prevent clotting; into this measure accurately 



6 Van Slyke and Cullen : Jour. Am. Med. Assn., Ixii, 155S, 1914. 



NEWER BLOOD CHEMISTRY. HI 

3.0 cubic centimeters of freshly drawn blood. For this purpose 
an accurately gTaduated pipette or hypodermic syringe may be 
used. After shaking up add 0.5 cubic centimeter of urease solu- 
tion (see page 299) and a few drops of amylic alcohol to pre- 
vent foaming. The tube is then stood aside for ten or fifteen 
minutes to allow the urease to act, after which 15.0 cubic 
centimeters of a saturated solution of potassium carbonate are 
added and the ammonia drawn off by a suction apparatus 
through another 100.0-cubic centimeter tube containing 15.0 
cubic centimeters of %oo normal hydrochloric acid (set-up of 
apparatus see Fig. 27). The absorption of ammonia is usually 
complete in from fifteen to twenty minutes, after which the re- 
maining acid is titrated with %oo normal sodium hydroxide 
solution, using methyl red or alizarin as the indicator. 

Calculations. — Each cubic centimeter of acid neutralized 
represents 0.01 gram of urea per 100.0 cubic centimeters of 
blood (1.0 percentage) or 0.0467 gram of urea nitrogen per 
100.0 cubic centimeters of blood. The amount of ammonia pres- 
ent in fresh blood is insufficient to interfere in this determina- 
tion, therefore, the number of cubic centimeters of acid neutral- 
ized by the ammonia resulting from the decomposition of the 
urea in 3 cubic centimeters of blood must be multiplied by 0.01 
to determine the percentage of blood urea present^ in the 
specimen. 

Rose and Colemen'^ suggests the colorimetric determina- 
tion of the ammonia, by using a standard ammonia solution and 
employing Nesslerization. F'or further details of this method 
see the original article or larger works on the subject. 

Betermination of Urea in Body Fluids and Tissues. — The 
above method is applicable without modification for the deter- 
mination -of the urea-content of other body fluids. Tissues first 
must be extracted with alcohol, the alcohol evaporated, and the 
residue dissolved in water before applying the method. In- 
hibitory substances may interfere with the action of the urease. 
This is especially true of quinine, quinol and formaldehyde. 

^ Rose and Colemen : Biochemical Bull., 3, 411, 1914. 



112 NEWER METHODS OF BLOOD EXAMINATION. 

D. NON-PROTEIN NITROGEN. 

Folin and Denis Colorimetric Method. — This method de- 
pends npon the removal of the protein from a small sample of 
blood by means of methyl alcohol, and the estimation of the 
nitrogen in the remaining solution by means of oxidation and 
Nesslerization. 

Technic. — ^Attach to a 2-cubic centimeter pipette a large 
hypodermic needle (B. D. and Co. L.2 g.22) by means of a 
short piece of rubber tubing and sterilize. Prepare the arm 
as for an intravenous injection and withdraw exactly 2 cubic 
centimeters of blood. Have previously prepared a 25-cubic 
centimeter flask by placing in it 20 cubic centimeters of pure 
(acetone free) methyl alcohol. As soon as the pipette is filled, 
remove the tube and needle from the pipette and blow the 
measured 2 cubic centimeters of blood into the flask and thor- 
oughly mix by shaking; fill the flask to the 25 cubic centimeter 
mark with methyl alcohol and again shake. Stand aside for 
two or three hours and filter through dry filter paper, add 3 
drops of a saturated alcoholic solution of zinc chloride to the 
filtrate and again filter through another dry filter paper. The 
second filtrate should have had all traces of coloring matter 
removed and should be perfectly clear and colorless. The filtrate 
thus obtained contains the non-protein nitrogen of the original 2 
cubic centimeters of blood. The nitrogen content of /the blood 
is now calculated by colorimetric methods as described in 
Chapter IX, page 311, and expressed in percentage or ^milli- 
grams per liter. 

Taylor and Hulton's Method. § — Place 10 cubic centimeters 
of a 3 to 1 mixture of absolute alcohol and ether in a small 
flask, stopper and weigh the flask and contents, remove stopper 
and allow 8 to 10 drops of blood from the finger of the patient to 
drop directly into the solution, replace the stopper and weigh 
the flask and contents. The increase in weight represents the 
weight of the blood. After a half-hour, filter the mixture into 
a digestion flask and wash the filter paper with 5 cubic centim- 
eters of alcohol-ether mixture. The filtrate which should be 
clear, is practically free from protein. Place the filtrate in a 

8 Taylor and Hulton : Jour. Biol. Chem., xxii, 63, 1915. 



NON-PROTEIN NITROGEN. 



113 



25-ciibic centimeter Kjeldalil flask (Fig. 28), add a small 
amount of potassium sulphate, several glass beads, and drive 
off tliQ alcoliol-ether mixture on a hot-plate or water-bath. 
When nearly dry add 1 cubic centimeter concentrated H2SO4 
and 300 to 400 milligrams of potassium, sulphate. Heat until 
the mixture is colorless. Transfer the contents to a 100-cubic 
centimeter flask, neutralize with 3 cubic centimeters of a 30- 
per cent. NaOH solution, fill the flask nearly full with am- 
monia-free water and add 5 cubic centimeters of Nessler- 




FiG. 28.— Kjeldahl Flasks— 1/0 Actual Size. 

Winkler solution (see Appendix), dilute to 100 cubic centimeter 
mark with ammonia-free water and place in colorimeter for 
comparison, with a standard ammonium, sulphate solution. 
Calculate! percentage in milligrams per liter as directed in 
Chapter IX, page 311. 



TOTAL NITROGEN. 

Kjeldahl Method. — The total nitrogen may be directly de- 
termined by the Kjeldahl method, 1 cubic centimeter of blood 
accurately measured is employed. 

The Folin-Farmer method may be employed with the fol- 
lowing preliminary modifications. Take 1 cubic centimeter of 



114 KEWER METHODS OF BLOOD EXAMINATION. 

blood, transfer to a 25-cubic centimeter flask containing dis- 
tilled water, dilute to the 25 cubic centimeter mark and take 1 
cubic centimeter of this diluted blood for the digestion and 
estimation outline on page 112. 



CHOLESTEROL CONTENT OF THE BLOOD. 

Gexeeal Coxsideratioxs. — Cholesterol was one of the first 
constituents of the blood to be determined colorimetrically. 
Grigaut was the first to describe (1910) a colorimetric method 
of estimating the cholesterol content of the blood. In the 
development of the color he made use of the Liebermann- 
Burchard reaction, and the technic of this part of the test is 
still carried out essentially as he originally described it. Two 
3'ears after the publication of Grigaut's method, Weston des- 
cribed a procedure in which the Salkowski color-reaction was 
employed. The Liebermann-Burchard reaction is the more satis- 
factory of the two. In 1913 Autenrieth and Funk described a 
slight modification of the Grigaut technic and adapted it to use 
with the Hellige colorimeter. 

Method of Myers.^ — One cubic centimeter of blood, plasma 
or serum is pipetted into a porcelain crucible or small beaker 
containing 4 to 5 gTams of plaster of Paris, stirred and dried, 
preferably in a dyring oven for an hour, emptied into a small 
paper extraction shell (4 centimeters long) and then inserted 
in a short glass tube (2.5x7 centimeters) in the bottom and 
sides of which are a number of small holes (Fig. 29). This is 
now attached to a large cork on a small reflux condenser and 
the tube and cork inserted in the neck of a 150-cubic centim- 
eter extraction flask containing about 20 to 25 cubic centim- 
eters of chloroform. Extraction is continued for 30 minutes, 
on an electric hot-plate, chloroform added to bring the volume 
up to 20 cubic centimeters, filtered and colorimetric estimation 
carried out as follows: 

Five cubic centimeters of the chloroform extract are 
pipetted into a dry test-tube, and 2 cubic centimeters of acetic 
anhydride and 0.1 cubic centimeter of concentrated sulphuric 



9 Myers and Wardell : Jour. Biol. Chem., 1918, xxxvi, 147. Myers, V. C. : 
Jour. Lab. and Clin. Med., Sept., 1920, v. 12, 776. 



CHOLESTEROL CONTENT OF THE BLOOD. 



115 



acid (best with 0.1 cubic centimeter pipette) are added. After 
thorough mixings the solution is placed in the dark for exactly 
ten minutes to allow the color to develop, and then compared 
with a standardized 0.005 per cent, aqueous solution of naphthol 




Fig. 29.— Cholesterol in blood-method of Myers Apparatus. 
(From Jour. Lab. and Clin. Med., No. 12, vol. v, p. 781.) 



green-B in a Bock-Benedict or Kober colorimeter. If the 
Duboscq colorimeter is used, it is necessary that the cups should 
be remounted in plaster of Paris, instead of balsam. 

With a good grade of acetic anhydride, it has been found 
that when an 0.005 per cent, solution of naphtliol green-B is 
used as a standard, and set at 15.5 millimeters on the Duboscq 
or Kober instrument 0.4 milligrams of cholesterol in 5 cubic 



116 NEWER METHODS OF BLOOD EXAMINATION. 

centimeters of chloroform treated with 2 cubic centimeters of 
acetic anhydride and 0.1 cubic centimeter of concentrated sul- 
phuric acid will read 15 millimeters. The color curve for 
both the cholesterol and naphthol green-B appear to fall in a 
straight line so that readings somewhat above or below the 
standard are accurate. 

If a cholesterol standard containing 0.4 milligrams to 5 
cubic centimeters or a naphthol green-B standard of ecjuivalent 
strength are employed^ the following formula may be used for 

S D 

the calculation : — X 0.0001 X — X 100 = cholesterol con- 

K 5 

tent of the blood in per cent.^ in which S stands for the depth 
of standard in millimeters^ R for the reading of the unknown, 
0.0004 the equivalent amount of cholesterol in 5 cubic centim- 
eters of chloroform, D the dilution of the chloroform extract 
from the 1 cubic centimeter otf blood, 5 the dilution of the 
standard and 100 the factor for 100 cubic centimeters. For 
example, 1 cubic centimeter of blood, 5 the dilution of the 
standard and 100 the factor for 100 cubic centimeters. For 

15 20 

example — X 0.0004 X — X 100 = 0.160 per cent. 
15 5 

Method of Autenrieth and Fiink.i<^ — Technic: Acqw- 
rately measure 2 cubic centimeters of blood, or blood serum, 
into a 100-cubic centimeter Erlenmeyer flask, and add 20 cubic 
centimeters of a 25 per cent, potassium hydrate solution. Heat 
on a water-bath for two hours, frequently shaking and adding 
water if necessary to maintain the volume, pour the residue into 
a separatory funnel and add 30 cubic centimeters of chloroform. 
Shake vigorously for five minutes and then separate; shake out 
with four more portions of chloroform of 20 cubic centimeters 
each, using the same procedure each time. The combined 
chloroform extracts, which will be brown or greenish in color, 
are then clarified by shaking with about 8 grams of anhydrous 
sodium sulphate and filtering. 

Dilute the filtrate to 100 cubic centimeters with chloroform ; 



10 Autenrieth and Funk : Miinch. med. Wochenschr., 1913, Ix, 1243. 



CHOLESTEROL CONTENT OF THE BLOOD. II7 

transfer 5 cubic centimeters of this extract to a small glass 
stoppered bottle of 10-cubic centimeter capacity, add 2 cubic 
centimeters of acetic anhydride and 0.1 cubic centimeter of con- 
centrated H2SO4 and agitate. Place in a water-bath in the 
dark at 35° C. for fifteen minutes. This will develop a green 
color. Coincidently a series of standards are prepared and 
treated in the same manner. The test is completed by com- 
paring the unknown with the known standards in a colorimeter. 

Preparation of Standards. — These are prepared by dis- 
solving in 100 cubic centimeters of chloroform, respectively (a) 
3.2, (b) 4.8, (c) 6.4, (d) 8.0, (e) 9.6 milligrams of pure 
cholesterol, and numbering them 1 to 5 respectively. In pre- 
paring the standards for comparison 5-cubic centimeter por- 
tions of each of the solutions are placed in small glass stoppered 
bottles and treated as was the unknown. Each standard then 
represents a concentration of 160 milligrams; 240 milligrams; 
320 milligrams; 400 milligrams and 480 milligrams respec- 
tively of cholesterol in 100 cubic centimeters of the original 
blood or serum. 

Normal blood contains 140 to 180 milligrams per 100 
grams of blood or about 0.15 per cent. 

1. The cholesterol content of the blood is lowered: 
(a) By a diet which is poor in lipoids. 

(o) By the occurrence of high temperature. 

2. The cholesterol content of the blood is increased: 

(a) By a diet excessively rich in lipoids. 

(b) By the presence of diseased conditions, especially 

diabetes, arteriosclerosis and nephritis. 

(c) During pregnancy. This lasts for a variable period 

after evacuation of the uterus. 

(d) By the presence of obstruction in the common bile 

duct. If the obstruction, however, is not abso- 
lute, as indicated by the degree of accompany- 
ing jaundice, the cholesterol content of the blood 
may not be increased. 



118 NEWER METHODS OF BLOOD EXAMINATION. 



ALKALI RESERVE OF BLOOD PLASMA. 



11 



The alkaline reserve of the blood plasma is maintained by 
the bicarbonates^ the alkali protein compounds and a small 
quantity of alkaU phosphates. Under normal conditions these 
substances are present in very constant quantities. A diminu- 
tion in the alkali reserve is known as acidosis and may be recog- 
nized clinically by a variety of symptoms and by characteristic 
alterations in the composition of the blood, the urine and the 
alveolar air. 

The alkali reserve maintains the plasma constantly at a 
slightly alkaline reaction, despite the fact that acid products of 
metabolism are being continually poured into the blood stream. 
The most important acid product from a quantitative standpoint 
is carbonic acid, this, as carbon dioxide, enters the plasma as it 
circulates through the tissues, being taken up partly in chemical 
combination and partly as dissolved carbonic acid. This results 
in an almost infinitesimal change in reaction in the direction 
of acidity. This slight change is sufficient to stimulate the 
respiratory center which in turn increases pulmonary ventilation, 
removes the excess of carbon dioxide and restores the normal 
alkaline reaction of the plasma. The greater the production of 
carbon dioxide in the tissues the greater the change in the re- 
action of the plasma, causing more powerful stimulation of the 
respiratory center, resulting in still more active pulmonary ven- 
tilation. This is the normal process which automatically tends 
to hasten the removal of CO2 from the blood and so prevents 
depletion of the alkali reserve. 

Pathologic conditions may develop, which permit the 
entrance of non- volatile acids (as betaoxybutyric) in addition to 
the CO2 into the plasma. This also stimulates the respiratory 
center and increases pulmonary ventilation, but the non-volatile 
acids are not given up in the lungs, so that the reduction of 
alkaline reserve is not so easily counteracted. This is of little 
importance unless there is so great an excess of fixed acids that 
the lungs are not able to maintain the carbon dioxide content 
of the blood at a low level, in such an event the reaction of the 



11 Marriott, W. McK. : Arch. Int. Med., June, 1916, xvii. Part 1, p. 840-851. 



ALIvALI RESERVE OF BLOOD PLASMA. 



119 



circulating plasma becomes less constantly alkaline until a point 
is reached which is incompatible with life. This point is ap- 
proximately neutrality. 

The reaction of the plasma is expressed in terms of the, 
negative logarithm of the hydrogen-ion concentration. (Ph). 
According to this method of notation a reaction of 7.0 is the 
expression of neutrality. The figures given in the diagram for 
reaction of the blood plasma are those obtained by the dialysis- 




I n izr 

Fig. 30.— Ph Concentration. 

indicator method described below, and closely approximates the 
true values (Fig. 30). 

In the columns the black areas represent the non- volatile 
acids, the shaded areas the carbon acid. Column I represents 
the plasma under normal conditions. The reaction P when 
measured under such circumstances that no carbonic acid is 
allowed to escape is approximately 7.5. If the carbonic acid is 
entirely removed, the reaction becomes more alkaline and 
reaches the point 8.5 ; this final reaction R may be considered 
the measure of the efi'ective alkali reserve. In the diagram the 
alkali reserve is represented by that part of the column reserve 
between the neutral point and 7.0 and the points marked 1\. 
As the neutral point at a given temperature does not vary, the 



120 NEWER METHODS OF BLOOD EXAMINATION. 

reaction at the point U, is the quantitative expression of the 
alkali reserve. 

Column II represents the condition of the plasma when 
a moderate degree of acidosis is present. The non-volatile 
acids are increased, but the carbonic acid is decreased so 
that the actual reaction P remains practically the same as under 
normal conditions. The alkali reserve, however, is depleted so 
that when the carbonic acid is removed the reaction R differs 
appreciably from the normal. 

Column III represents the condition of the plasma, when 
the alkali reserve is so greatly depleted that the diminished 
amount of carbon dioxide can no longer compensate, as in 
Column II, therefore, an actual change in the reaction of the 
plasma, as it exists in the vessels, occurs. On removing carbon 
dioxide the reaction differs very greatly from the normal. The 
length of the shaded portions of the columns indicate, in a gen- 
eral way, the variations of the tension of the carbon dioxide in 
the plasma and hence in the alveolar air, which is presumably 
in equilibrium with the plasma. 

Provided the respiratory center does not vary in its excita- 
bility the reaction of the circulatory plasma is maintained at a 
constant point, except when a very marked acidosis occurs. The 
tension of the carbon dioxide in the alveolar air generally bears 
a constant relation to the alkali reserve since it is only in severe 
acidosis that changes occur in the reaction of the plasma as it 
exists in the body. The measurement of the reaction, the hydro- 
gen-ion concentration is a measure of the excitability of the 
respiratory center rather than of the degree of acidosis. 

The measurement may be most accurately made by the 
electrometric gas-chain method, a somewhat difficult procedure, 
and one not readily adapted to clinical work. As a clinically 
accurate chemical method Levy, Eowntree, and Marriott ^^ j^aye 
devised a plan of determining the reaction of the plasma by 
means of an indicator. The method consists of dialyzing serum 
or whole blood against salt solution in order to remove the 
coloring matters and the proteins. The hydrogen-ion concen- 
tration of the dialyzate is determined by means of phenolsul- 



12 Levy, Rowntree and Marriott; Arch. Int. Med., 1915, xvi, 



HYDROGEN-ION CONCENTRATION OF BLOOD. 121 

pliouphtlialein as an indicator, phosphate solutions of known 
Ph concentration being used as standards for comparison. 

Peocedure.i^ — The work mnst be done in a room free from 
the fnmes of acids or ammonia. Three cubic centimeters of 
blood or serum is run by means of a blunt pipette into a dialyz- 
ing sac, previously washed inside and out with 0.8 per cent, 
sodium chloride. The sac containing the test-fluid is then 
lowered into a small, test-tube (slightly larger in diameter than 
the sac) containing exactly 3 cubic centimeters salt solution 
(0.8 per cent.) until the fluid levels coincide. Five minutes 
later the collodion sac is removed and 5 drops of an aqueous 
0.01 per cent, solution of phenolsulphonephthalein added and 




Fig. 30a.— Hydrogen-ion Concentration Colorimeter. 

thoroughly mixed with the salt solution in the test-tube (the 
dialyzate). The tube is then compared with the standards (see 
note) until the corresponding color is found. This indicates 
the hydrogen-ion concentration present in the dialyzate. 

Significance. — ^A rise in the hydrogen-ion concentration 
of the blood is significant of acidosis because it indicates a fail- 
ure on the part of the protective mechanism of the body to 
preserve its proper reaction. 

Preparation of Bialyzing Sacs. — Dissolve 1 ounce of col- 
lodion in 500 cubic centimeters of an equal mixture of ethyl 
alcohol and ether. This solution should not be used for sev- 
eral days after making and any sediment formed should be 
removed by pouring the clear solution into another vessel. 
Protect carefully from evaporation. A small test-tube is filled 
with the mixture ; then half the contents poured out, the remain- 
is Levy, Rowntree and Marriott : Jour. A. M. A., Ixv, 14, 1915 and Arch. 
Int. Med., xvi, 3, 390, 1915. 



132 NEWER METHODS OF BLOOD EXAMINATION. 

ing contents is allowed to flow into the bottom of the tube, then 
again inverted while being rotated on its long axis, as the re- 
maining collodion is drained off. Care should be exercised in the 
manipulation to secure a uniform layer of collodion within the 
tube. Clamp the tube in the inverted position until all odor 
of ether disappears (10 to 20 minutes), rinse the inside of the 
tube with several changes of cold water, loosen the collodion 
around the rim of the tube with a sharp knife and then insinuate 
cold water between the collodion skin and the tube by means of 
a pipette. By careful manipulation and by gentle traction the 
sac may be removed unbroken from the tube. Several sacs may 
be prepared at one time and preserved in cold water for use. 

Preparation of the Salt Solution. — C.P. sodium chloride 
only should be used in the 0.8 per cent, salt solution. The 
presence of acids other than carbonic will . interfere with the 
test. The addition of a few drops of '^^phthalein indicator" 
should produce a yellowish tinge, which on boiling should be- 
come slightly brownish. If on addition of the indicator a pink 
color develops, then the solution is alkaline and cannot be used. 

Preparation of Standard Colors. — Standard phosphate mix- 
tures (method of Sorensen). Prepare two solutions: (a) 
potassium di-hydrogen phosphate (KH2PO4) and (&) di- 
sodium hydrogen phosphate N'a2HP04+2H20, which is pre- 
pared by exposing fresh Na2HP044-12H20 for two weeks to 
the air protected from dust. 

(a) 1/15 mol. potassium di-hydrogen phosphate or primary 
(acid) potassium phosphate solution is prepared by dissolving 
9.078 gTams of pure crystallized salt in 1 liter of fresh dis- 
tilled water. 

(h) 1/15 mol. di-sodium hydrogen phosphate or secondary 
(alkaline) sodium phosphate is prepared by dissolving 11.876 
grams of the desiccated chemically pure salt in 1 liter of freshly 
distilled water. This solution should give a strong alkaline 
reaction to the "phthalein indicator." 

The solutions are mixed in the proportions indicated in 
the table to obtain the desired Ph (see Table). 

Three cubic centimeters of each of the phosphate mixture 
solutions are placed in suitable small test-tubes (100 x 10 milli- 



HYDROGEN-ION CONCENTRATION OF BLOOD. 



123 



meters inside measurement) 5 drops of an aqueous 0.01 per 
cent, solution of phenolsulphonphthalein are added to each tube, 
the tops of which are then sealed off. The series of color tubes, 
each marked with, its Ph equivalent constitutes the standards 
for comparison of color in carrying out the determination. It 
should be remembered that the colors may fade slightly after 
a month's time but may still be used for comparison if less 
indicator is added to the ^^mknown.'^ 

Comparison of Unknown with Standards. — For this a good 
natural or artificial light and a dead white background are 
essential. Eeadings must be made immediately after dialyza- 
tion. The two tubes most nearly approximating the color of 
the specimen are selected and one placed on each side of the 
specimen. These are critically inspected against the white back- 
ground, reversing the relative positions of the tubes will aid in 
differentiating differences in color. It must be borne in mind 
that in comparing the ^^unknown'^ with the standard colors it is 
matching the quality, not the intensity of color, as in ordinary 
colorimetry, that makes for accuracy. 

Table for Preparation of Standard Colors. 



pH 


6.4 fi fi fi-« 


7.0 


7.1 


7 '>, 


7 3 


7.4 


7.5 


7.6 


7.7 


7.8 


8.0 


8.2 


8 1 
















Primary potassium 
Pfiospfiatec.c. 


73 


63 


61 


37 


32 


27 


23 


19 


15.8 


13.2 


11.0 


8.8 


5.6 


3.2 


2.0 


Secondary sodium 
Phosphate c.c. 


27 


. ... 
37 


49 


63 


68 


73 


77 


81 


84.2 86.8 


89.0 


91.2 


94.4 


96.8 


89.0 



Significance of the Test. — ^Experimental study has shown 
that under normal conditions the hydrogen-ion concentration 
of the whole blood, or of the serum is not subject to great varia- 
tion, while in a great variety of disease conditions it is in no 
way affected. Oxalated blood from normal individuals gives a 
dialyzate with a Ph, varying from 7.4 to 7.6 while that of the 
serum ranges between 7.6 and 7.8. Variations from these figures 
toward the acid side are, as far as we know, met only in con- 
ditions which are from both the clinical and laboratory stand- 
point evidenced by acidosis. In clinical acidosis the Ph of 



124 NEWER METHODS OF BLOOD EXAMINATION. 

oxalated blood will be found between 7.3 and 7.1 and that of the 
plasma from 7.55 to 7.3. 

Note. — ^A series of prepared color standards may be ob- 
tained from any surgical or chemical supply house or directly 
from the manufacturers, Hynson, Westcott and Dunning, of 
Baltimore, Md. (See Fig. 31). 

DETERMINATION OF ALVEOLAR CARBON- 
DIOXIDE TENSION.^^ 

The effective concentration of carbon dioxide in the alveolar 
air is diminished in acidosis. By determining this tension (con- 
centration) a diagnosis of acidosis may be made, the degree of 
severity estimated and the results of treatment followed. The 
method of study divides its,elf into (a) the collection of the 
alveolar air and (6) the analysis of the sample. 

Collection of the Alveolar Air. — A rubber-bag having 
about 1500 cubic centimeters capacity (basket-ball or foot-ball 
bladder), is, connected by means of a short rubber tube with a 
glass mouth-piece. About 600 cubic centimeters of air are 
blown into the bag by an atomizer-bulb, and retained by a 
pinch-cock. The subject to be tested sl-'ould be at rest and 
breathing naturally. At the end of a normal expiration the 
subject takes the tube in his mouth, the pinch-cock is released, 
and the subject's nose closed mechanically. The subject breathes 
back and forth four times in twenty seconds, emptying the 
bag at each inspiration. The subject's breathing should be 
directed by. the observer, which is greatly facilitated by the em- 
ployment of a stop-watch, although more frequent breathing will 
not materially alter the result. At the end of twenty seconds the 
tube is clamped off and the air analyzed. The analysis should 
be carried out within three minutes time as carbon dioxide 
rapidly escapes through the rubber. In comatose patients some 
difficulty will be experienced in collecting the sample. Here 
the initial amount of air should be over 1000 cubic centim- 
eters, and the period of rebreathing prolonged to thirty seconds. 
The aim in rebreathing is to empty the bag as nearly as pos- 
sible with each inspiration in order that the CO2 tension in 



14 Marriott, W. McK. : Jour. A. M. A., 1916, May 20, Ixvi, 22, 1594. 



ALVEOLAR CARBON-DIOXIDE TENSION. 



125 



the bag may rapidly balance that in the lungs. In comatose 
patients., a gas anesthetic mask may be used. 

For analysis of the air samples, eight test-tubes are re- 
quired, each containing standard phosphate solution; a standard 
carbonate solution; a small test-tube; a glass tube or pipette 
drawn out into a capillary point and a box of samples for color 
comparison (Fig. 30a). 

The method depends' upon the fact that if a current of air 
containing COo is passed through a solution of sodium car- 




FiG. 31.— MARRIOTT Alveolar Air Testing Outfit. 



bonate until the solution is saturated, the final solution will 
contain sodium bicarbonate and dissolved CO2. The reaction of 
such a solution will depend on the relative amounts of the alka- 
line bicarbonate and the acid carbon dioxide present. This, in 
turn will depend upon the tension of the CO2 in the air with 
which the mixture has been saturated and will he independent 
of the volume of air hlown througli, provided saturation has 
been once obtained. Higli tensions of CO2 change the reaction 
toward the acid side, low tensions have the reverse effect; hence 
the reaction of such a solution is a measure of the tension of 
the CO2 in the air with which it has been saturated. The re- 
action of such a solution may be determined by adding to it a 
suitable indicator ( phenol sulphonphthalein) which shows over 



126 NEWER METHODS OF BLOOD EXAMINATION. 

a considerable range of reaction definite color changes. A cer- 
tain change of color indicates a certain reaction. 

Solutions of given reaction may be prepared by mixing acid 
and alkaline phosphates in definite proportions (see Appendix 
page 501). Such solutions may be kept unalterated for long 
periods of time and serve as standards for comparison. 

Proportions in Which Solutions are Mixed. 

Tension in mm 10 15 20 25 30 35 40 45 

Acid potassium phos- 
phate c.c 17.8 25.2 31.0 35.7 40.5 45.0 47.0 50.2 

Alkaline sodium phos- 
phate c.c 82.2 74.8 69.0 64.3 59.5 55.0 53.0 49.8 

The solutions thus made up are put in small test-tubes 
(10 X 75 millimeters convenient) and stoppered or better, 
sealed off. These standard test-tubes should be kept in the dark 
when not in use. 

The standard bicarbonate solution is prepared either by 
weighing out 0.530 gram of desiccated sodium, carbonate, or by 
measuring accurately 100.0 cubic centimeters of N/10 sodium 
hydroxide into a 100-cubic centimeter volumetric flask, adding 
200.0 cubic centimeters of a 0.01 per cent, phenolsulphonphtha- 
lein solution and diluting the whole up to the 1000.0 cubic 
centimeter mark with distilled water. 

Carbon dioxide from a cylinder or from the lungs may then 
be passed through this solution to convert the hydroxide to car- 
bonate, or the solution may simply be used as it is, as the 
alveolar air which will be blown through the solution, subse- 
quently will accomplish the same purpose. 

The comparison with the standard colors is conveniently 
made in a box similar toi that used in the Sahli hemoglobinom- 
eter, but containing three instead of two compartments. By the 
use of this device slight color changes may be readily detected 
and the temperature of the tubes unaffected by the heat of the 
hands. 

A standard temperature may be employed by immersing 
tubes and rack in water at a given temperature. 

All the standard solutions used in this method should be 
kept in glass, which does not readily give off alkali such as 



ALVEOLAR CARBON-DIOXIDE TENSION. 12? 

"Jena/^ or Pyrex glass. A small amount of thymol or toluene 
may be added to prevent the development of molds. 

Technic. — To analyze a sample of air, 2 or 3 cubic centim- 
eters of the standard bicarbonate solution are poured into a 
clean test-tube, of the same diameter as the tubes containing the 
standard color solutions, but from 100 to 150 millimeters long. 
Air from the bag is then blown through the solution by means 
of a tube drawn out to a fine capillary point, until the solution 
is saturated, as shown by the fact that no further color change 
occurs. (If the operator first blows his own breath through the 
solution so as to bring it up to approximate equilibrium with 
the alveolar air, saturation may then be accomplished with as 
little as 100 cubic centimeters of air from the bag blown through 
during thirty seconds.) The same bicarbonate solution may be 
used for repeated determinations. The tube is then stoppered 
and the color immediately compared with that in the standard 
tubes. By interpolation one can readily read to millimeters. 
Color changes are not quite so sharp above 35 millimeters as 
at the lower end of the scale but here the changes are of less 
significance. In making the color comparisons the solution 
being compared is placed between the two standards which it 
most nearly matches, changing the location of the standard 
tubes in the test-holder until there is no doubt about the rela- 
tionship of the solution tested to the standards. 

The standard solutions described are so prepared as to 
give correct results when the determination is carried out at a 
temperature of from 20° to 25° C. (68° to 77° F.) . Best results 
are obtained when the tubes are immersed in water at approxi- 
mately 25° C. during the blowing. This is however, not impera- 
tive as the differences in reading owing to changes of tempera- 
ture are under ordinary circumstances negligible. Barometric 
variations and variations in the temperature of the subject are 
unimportant, less than 1 millimeter. 

Caution. — Duplicate determinations including the collec- 
tion of the air sample should be made. If the technic is accu- 
rate the duplicate determinations will usually agree within 2 
millimeters. Errors in technic in collecting the sample lead to 
low rather than to too high results. 



128 NEWER METHODS OF BLOOD EXAMINATION. 

Normal Finding. — ^In normal adults at rest, the COo 
tension in the alveolar air determined by the method of Marriott 
varies from 40 to 45 millimeters. Tensions between 30 and 35 
millimeters are indicative of a mild degree of acidosis. When 
the tension is as low as 20 millimeters the individual may be 
considered in imminent danger. In coma associated with 
acidosis the tension may be as low as from 8 to 10 millimeters. 

In infants the normal tension is from 3 to 5 millimeters 
lower than in adults. 

Conditions other than acidosis may affect the CO2 tension. 
Lowered alveolar CO2 tension follows stimulation of the respira- 
tory center. This may be brought about by administration of 
caffeine and possibly by intracranial lesions. Increased alveolar 
CO2 tension follows depression of the respiratory center, as 
after the administration of morphine and as a result of certain 
infections. 

Method of Collecting Air from Infants. — A special mask 
for this purpose as described by Howland and Marriott ^^ may 
be made as follows : A sheet of thin rubber tissue (dental rubber 
dam) 8 x 10 inches is perforated in the center by a piece of hot 
metal or glass tubing of large bore. The hole is stretched over 
a hygeia-nipple and slipped down to the lower rim, where it is 
sealed with a small amount of auto tube repair cement. A slip 
of adhesive plaster % inch wide is applied around the rim of 
the nipple so as to overlap the rubber tissue and hold; it firmly 
in place. Finally, the extreme end of the nipple is cut off and 
a short glass ,tube % inch in diameter inserted. In making the 
collection of alveolar air from infants a rubber bag of 500 cubic 
centimeters capacity is partially filled with an atomizer bulb, the 
tube pinched off and its end joined to the mask by a short piece 
of rubber tubing. Crying for at least one minute before collec- 
tion should be avoided, rebreathing should be begun just at the 
end of expiration if possible and continued for 30 seconds. Cry- 
ing during rebreathing usually occurs but unless very vigorous 
does not materially affect the reading. The initial amount of air 
in the bag should be such that during respiration the bag is from 
one-half to two-thirds empty but never completely collapsed. 



15 Howland and Marriott: Amer. Jour. Dis. of Child., May, 1916. 



BLOOD SUGAR. 



129 



The amount of air required for infants under 1 year of age 
varies from 250 to 400 cubic centimeters. 

BLOOD SUGAR. 

Method of Epstein.i^ — Peinciple: This method is a modi- 
fication of the Lewis and Benedict procedure;, being based on 
the same principle but making possible the determination of 
reducing sugar in finger blood (0.1-0.2 cubic centimeter) with a 
sufficient degree of accuracy for clinical purposes^ and with 
little expediture of time. The determination is a colorimetric 
one in which the Duboscq, the Sahli-Gower or the Hillege colo- 
rimeter may be used. 





Fig. 32.— Epstein Apparatus for Blood Sugar, 



Procedure. — The apparatus!'^ shown in the illustration 
(Fig. 32) and the following reagents are necessary: 

1. Picric acid, saturated solution. 

2. Sodium carbonate, 10 per cent, solution. 

3. Sodium fluorid or potassium oxalate, 2 per cent, 
solution. 

Pour 1 or 2 drops of the fluorid or oxalate solution into the 
graduated test tube (Fig. 32). By means of the blood pipette, 



16 Epstein : Jour. Amer. Med. Assn., 1914, 63, 1067. 

17 The tubes belonging to this hemoglobinometer are not aU equally cali- 
brated. With sonae the 50 per cent, mark represents a volume of 1.0 cubic 
centimeter; with others 1.0 cubic centimeter of fluid reaches up to the 43, 
4.5, 46 or 47 per cent. mark. The error in the calibration is generally below 
the 10 per cent, mark; the graduations above this mark are usually correct. 
By means of the standard 1.0 cubic centimeter pipette one can readily deter- 
mine whether or not a given tube is properly calibrated. In order to facilitate 
a correct reading of the percentage of sugar in these hemoglobinometer tubes, 
it is essential to have 1.0 cubic centimeter of fluid stand at 50. To overcome 
a discrepancy (if any exists) in the calibration of a given tube, one may put 
one, two, or three small glass beads in the bottom of the tube, of such size as 
to raise the meniscus of 1.0 cubic centimeter of fluid up to the 50 per cent, 
mark. 

9 



130 NEWER J^IETHODS OF BLOOD EXAMINATION. 

0.2 cubic centimeter of blood is obtained from the tip of the 
finger or the lobe of the ear and is discharged into the tube 
containing the fluorid solution. The pipette is rinsed two or 
three times with distilled water and the Avashings added to the 
blood in the tube. Distilled water is then added to the 1.0 cubic 
centimeter mark. After laking has taken place, picric acid is 
then added, a few drops at a time, up to the 2.5 cubic centimeter 
mark, shaking the tube gently with each addition of the acid. 
Precipitation of the blood-proteins takes place; the sugar, 
together with an excess of picric acid sufficient for the reaction, 
stays in solution. The tube is finally shaken vigorously (cover- 
ing the end of the tube with the finger) and the contents filtered 
through a small filter, or, better still, centrifugated for one or 
two minutes. 

One cubic centimeter of the filtrate or of the clear super- 
natant fluid obtained on centrifugalization is withdrawn, put 
into the plain test-tube and heated carefully over the naked 
flame. The contents of the tube are boiled until all but 2 or 3 
drops of the solution has evaporated. One-half cubic centim- 
eter of the 10 per cent, sodium carbonate solution is then added 
and the tube heated again until the contents are concentrated 
to a small volume equal to about 2 or 3 drops. The color of the 
fluid changes from yellow to deep or reddish brown and the 
reaction is completed. 

Three or four drops of distilled water are added and the 
tube warmed gently. The contents are then transferred to the 
graduated tube of the hemoglobinometer. The boiling tube is 
rinsed several times with water (using only 3 or 4 drops at a 
time). The tube is warmed with each rinsing before trans- 
ferring the contents to the gTaduated tube. The volume of 
fluid is then made up to the mark 50 on the scale. 

The color of the resulting solution is compared with that 
of the two standard tubes, A and B, which accompany the 
instrument. 

If it is darker than standard A (representing 0.05 per 
cent, of sugar) and lighter than standard B (representing 0.1 
per cent.) the first standard is used for comparison. In either 
case the solution in the graduated tube is diluted gradually 



BLOOD SUGAK. 131 

with water (just as is usually done in hemoglobin estimations) 
until the colors match. 

The percentage of sugar in the blood is then computed thus : 
Using the lighter standard A, the figure of the scale, divided by 
1000 represents the percentage of sugar in the blood. For 
example, if the tube reads 86 ; then the result is 

86 

=0.086 per cent. 

1000 

When the standard B is used for comparison, the figure on 
the scale is multiplied by 2 and divided by 1000. For example, 
if the tube reads 73 ; then the percentage of sugar is 

73 X 2 

= 0.146 per cent. 

1000 

With the instructions given the above formulas may be used 
for direct computation of the percentage of sugar, only, when 
0.2 cubic centimeter of blood is used in the determination 
When, however, only 0.1 cubic centimeter of blood is used, the 
formulas apply as well, but the value obtained must be 
multiplied by 2. 

It is better, in cases in which a high sugar content in the 
blood is suspected (in diabetes for example) to use only 0.1 
cubic centimeter of blood for the determination. In all other 
cases 0.2 cubic centimeter of blood should be used. 

Method of Lewis and Benedict.i^ — Prin^ciple: The red 
color obtained by heating a glucose solution with picric acid 
and sodium carbonate is employed as the basis of this colori- 
metric determination. The blood protein is removed by pre- 
cipitation with picric acid. 

Peocedure. — A little more than 2 cubic centimeters of 
blood are drawn into an all glass hypodermic syringe through an 
ordinary hypodermic needle. After the needle is disconnected, 
the blood is allowed to flow back to the 2.0 cubic centimeter 
mark and the contents of the syringe is discharged directly into 
a 25.0-cubic centimeter volumetric flask containing 5.0 cubic 



18 Lewis and Benedict: Jour. Biol. Chem., 1915, xx, 61; also Myers and 
Bailey: Jour. Biol. Chem., 1916, xxiv, 147; also Hawk: Physiologic Chemistry,. 
Blakiston, 1916. 



132 NEWER METHODS OF BLOOD EXAMINATION. 

centimeters of water. The contents of the flask are shaken to 
insure thorough mixing and laking of the blood. To this is 
then added 15 cubic centimeters of saturated aqueous solution 
of picric acid and 3 or 4 drops of alcohol to prevent foaming; 
the contents of the flask is then made up to the 25.0 cubic 
centimeter mark with water and well shaken. After filtration 8 
cubic centimeters of aliquots are measured out into large Jena 
test tubes for duplicate determination. Two cubic centimeters 
of saturated picric acid solution and exactly 1 cubic centimeter 
of 10 per cent, sodium carbonate are added (as well as 2 glass 
beads and 2 or 3 drops of mineral oil) and the contents of the 
tube are evaporated rapidly over a direct flame until precipita- 
tion occurs. About 3 cubic centimeters of water are next added 
and the tube again heated to boiling to dissolve the precipitate ; 
the contents of the tube are then transferred to a 10-cubic 
centimeter volumetric flask together with two washings and 
diluted to the 10.0 cubic centimeter mark. (In case of hyper- 
glycemia the final volume of the fiuid is nlade 25 or 50 cubic 
centimeters, and the results accordingly multiplied by 2.5 or 
5.0) cooled, shaken and then filtered through cotton into 
the chamber of a Duboscq colorimeter (see Fig. 69). The 
color is compared at once with that obtained from 0.64 
milligrams of glucose, 5 cubic centimeters of saturated picric 
acid, and 1 cubic centimeter of 19 per cent, sodium carbonate, 
after evaporation to precipitation over a free flame and dilution 
to 10 cubic centimeters as was the unknown, or against the pic- 
ramic acid standard mentioned in Appendix, page 503. 

Calcuiation'. — If directions are followed exactly the cal- 
culation is as follows: 

,^.,T , 1 reading of standard f Milligrams 
MiUiCTams glucose . . \ ^ i - 

° ° ^ — X < of glucose m 

reading of unknown I standard 

Pearce's Hodification of Lewis-Benedict Method. ^^ — The 

modification entails the use of an autoclave instead of the free 
flame and has the advantage of decreasing danger of loss, at the 
same time making it possible to carry out a large number of 
estimations simultaneously. 



in unknown [ 



19 Pearce : Jour. Biol. Chem., 1915, xxii, 525. 



BLOOD SUGAR. 133 

Proceed exactly as in the Lewis-Benedict, but use 6 
cubic centimeters of the picric acid filtrate instead of 8 cubic 
centimeters and instead of heating over the free flame introduce 
into an autoclave for fifteen to thirty minutes at about 20 
pounds pressure to the square inch. Compare with standard in 
a colorimeter. The standard, recommended by Lewis and Bene- 
dict may be diluted one-fouth or allowed for by calculation, 
since 6 cubic centimeters of filtrate are used in place of 8 cubic 
centimeters. 

Normal human blood contains slightly less than 0.1 per 
cent, of glucose. Strouse^o places the average at 0.085 per 
cent. It is slightly increased (up to 0.18 per cent.) after a 
meal. In cases of glycosuria the hyperglycemia may reach from 
0.3 to 1.0 per cent. 



20Strouse: Bull. Johns Hop. Hosp., 1915, xxvi, 211. 



VI. 

SPHYGMOMANOMETRY AND 
SPHYGMOGRAPHY. 



A. SPHYGMOMANOMETRY. 

Capillary Blood-pressure. — ^The pressure of the blood in the 
capillaries is low, because of the resistance offered to the progress 
of the blood by the fine bore of the vessels, and because of the 
relatively large cross-sectional area of all the capillaries com- 
pared to that of the aorta and great vessels. 

The capillary pressure has been found to be much lower 
than in the arteries, and considerably higher than the pressure 
in the great veins. This pressure has been found to equal that 
required to sustain a column of from 24 to 54 millimeters of 
mercury. 1 Clinically the capillary blood-pressure is not of 
sufficient importance to warrant a further discussion here. 

Terms and Definitions. — The Pulse: The pulse is the 
rhythmically recurring impulse propagated by the systole of the 
left ventricle and palpable throughout the arterial system. 

Arterial Pressure. — By arterial pressure is meant the 
degree of force exerted by the blood within the vessel. It is 
primarily dependent on the strength of the heart as measured 
by its rate and by the volume of blood expelled at each systole, 
balanced by the elasticity of the vessel walls and capillary 
resistance. 

The Systolic Pressure (Fig. 33). — The systolic pressure, 
as indicated by the sphygmomanometer, represents the pressure 
within the vessels at the time of systole of the ventricles. 

The Diastolic Pressure (Fig. 33). — The diastolic pres- 
sure represents the ebb to which the arterial pressure falls during 
cardiac diastole. 

The Pulse-pressure, Ean-ge or Amplitude (Fig. 33). 
— The arterial pulse is caused by variations in pressure within 



1 Amer. Textbook of Physiology, p. 377. 

(134) 



SPHYGMOMANOMETRY. 



135 



the arterial system caused by the intermittent pumping action of 
the heart. The difference between systolic and diastolic pressures, 
i.e., the variation in pressure occurring within the vessel during 
a complete cardiac cycle, is termed the pulse-pressure. This 
figure is obtained by subtracting the diastolic from the systolic 
pressure. The normal pulse-rate ranges between 35 and 45 
millimeters of mercury. 

Variations in the pulse-pressure in the same individual 
constitute a most important part of the study of blood-pressure. 

It is theoretically possible that the pulse-pressure should be 
influenced in at least three ways : 1. An increase in the amount 
of blood delivered at each beat of the heart would tend to increase 
the difference between systolic and diastolic pressures. 2. A 



Systolic=120 

Pulse- 
pressure=:40 

Diastolic=:80 




Mean=100 



Fig. 33.— Normal Pulse Tracing : Showing Relation op Systolic, Dias- 
tolic, Pulse-pressure and Mean. Pulse-pressure Equals 30. 



rapid emptying of the vessels, the cardiac output remaining the 
same, would tend to increase this difference. This would occur 
independently of whether the blood was passed onward into the 
capillaries or was regurgitated into the ventricle. 3. Eigid 
vessel-walls would increase pulse-pressure. If the arteries were 
rigid tubes, the heart at each systole would be compelled to move 
the blood in the arterial system as a whole, while during diastole 
the flow would cease. There would thus be an increase of 
pressure during systole, while during diastole it must fall rapidly 
toward zero. 

The Mean Pressuke. — ^The mean blood-pressure is valuable 
chiefly as an indication of the amount of strain to which the 
heart and larger vessels are subjected. It varies with the pulse- 
pressure, the systolic pressure, and the diastolic pressure. 

To obtain the mean pressure, divide the sum of the systolic 
and diastolic pressures by two, or add half of the pulse-pressure 
to the diastolic pressure (Fig. 33). 



136 SPHYGMOMANOMETRY AND SPHYGMOGRAPHY. 

Pathologically, the pulse-pressure increases in organic 
diseases of the kidneys, in arteriosclerosis, and in aortic insuffi- 
ciency. It diminishes from other organic diseases of the heart, 
affecting the valves or myocardium.^ 

Significance of Pulse-pressure. — The normal pulse-pressure 
indicates a normally acting heart, a proper systolic output, and 
a normal vascular distribution of blood. Any condition which 
interferes with the normal flow of blood from the capillaries 
will tend to increase the systolic pressure, and will produce a 
greater pulse-pressure. We find therefore an increased pulse- 
pressure in arteriosclerosis. The increased systolic output of 
the large heart in aortic regurgitation produces, during com- 
pensation, a very large pulse-pressure. According to Gerhardt, 
the systolic blood-pressure in broken compensation may be high, 
but the pulse-pressure is always diminished becoming greater 
as compensation is re-established. An increased pulse-pressure 
in the absence of any other explanation is very suggestive of 
organic disease of the kidneys or arteriosclerosis. Myocardial de- 
generation of all kinds is productive of a reduction of the pulse- 
pressure, and the mean of this is a fair estimation of the state 
of the myocardium, upon which is based the work test of 
G-ralipner (test on page 152). 

The Principle of the Sphygmomanoineter. — Vital tissue is 
perfectly elastic. Therefore any pressure applied to the surface 
of the body will be directly transmitted to the underlying 
structures without loss of force. It is upon this principle that 
the indirect method of measuring the blood-pressure is based. 

Pressure is applied to an accessible part of the body over a 
large blood-vessel, such as the brachial. If the amount of this 
pressure is sufficient to overcome the pressure of the blood within 
the vessel, the vessel will be collapsed and the pulse prevented 
from passing beyond it. If the amount of the compressing force 
is measured and expressed in definite terms of weight (as 
millimeters of a column of mercury) then we can, by applying 
just sufficient pressure to collapse the vessel, measure the amount 
of force exerted by the blood in preventing this collapse. 

In practice the compressing force is obtained by a cautery- 
bulb or a small hand-pump, and applied to the arm by means of 

2 Eichberg : Jour. Amer. Med, Assoc, Sept. 19, 1908. 



PLATE IV. 




Sphygmomanometer in Position for Observation. 



SPHYGMOMANOMETRY. 



137 



a hollow, flat rubber bag. This is applied about the arm and 
held there by some form of inelastic cuff. A tube communicating 
with a mercury or an aneroid manometer measures the amount 
of pressure applied over the vessel. 

The Sphygmomanometer. — The apparatus devised for meas- 
uring blood-pressure may be divided into three classes: 1. The 
spring. 2. The mercury. 3. The aneroid. 

1. The spring type of instrument represents efforts to pro- 




FiG. 34.— Faught's Mercury Sphygmomanometer. 



duce a clinical instrument of small size and minimum cost; so 
far these have not measured up to the requirements of clinical 
medicine, and, therefore, cannot be recommended for clinical 
use, as they are neither dependable nor accurate. 

2. The mercury type of apparatus is divided into two 
classes: One of these employs a vertical tube into which the 
mercury column is forced from a large containing chamber in 
the base of the instrument. The pressure is measured in milli- 
meters of mercury on an appropriate scale attached to the verti- 
cal glass tube. 

The other employs a glass tube (similar to that first used by 
Poiseuille) bent in the form of a "U'' with the open ends up. 
This tube is partly filled with mercury and one end connected 



138 SPHYGMOMANOMETRY AND SPHYGMOGRAPHY. 

by means of suitable tubing with the compression part of the 
apparatus. The pressure is measured upon a suitable scale placed 
between the two limbs of the tube, and is represented by the 
difference in the height of the mercury in the two limbs of the 
"U" tube. 

The mercury sphygmomanometer (Fig. 34) bearing the 
author^s name is modeled after the type of apparatus employing 
the "U" tube and is designed to overcome the many shortcomings 
of the earlier instruments and to furnish an instrument which is 




Fig. 35.— Faught's Clinical Sphygmomanometer. 

easy to use, difficult to get out of order, accurate and as light 
and portable as is compatible with exactness and strength. 

The mahogany case, which encloses the complete apparatus, 
including the arm-band and pump, measures 4 x 4% x 16 inches 
and weighs 3 pounds and 9 ounces. The lid is hinged at one end 
and when raised supports the working parts of the apparatus. 
A spring check allows the lid to be raised to a vertical position, 
where it is automatically held locked during observation. 

The "U^^ tube is provided with a scale, which has been 
arranged to give the reading directly in millimeters of mercury, 
each space representing 2 millimeters Hg. The range is from 
to 300. 

A special and distinctive feature of the apparatus is the 
means of preventing loss of mercury from the manometer tube 
when the instrument is not in use. This is accomplished by 



SPHYGMOMANOMETRY. 



139 



means of two small cocks placed at either extremity of the "U^' 
tube, and which are kept closed when the apparatus is not in use. 
By eliminating all detachable parts, the time required to 
make the reading is reduced to a minimum, the only prelimi- 
naries to the test being to lift the lid, open three cocks and 
attach two tubes to their respective nipples. 




Fig. 36.— Actual Size Pocket Indicator, 



3. The aneroid sphygmomanometer represents the acme of 
pressure-measuring instruments 3^et made. This instrument is 
at once small, compact, reliable, and accurate, and, when 
properly used, almost indestructible. 

The principle is that of the aneroid barometer except that 
in the sphygmomanometer of this type the fixed pressure within 
the chambers (see Fig. 37) is the atmospheric pressure (instead 
of a vacuum, as in the barometer), while the variable pressure is 
that produced within the apparatus by means of the pump and 
which can be changed at the will of the operator. As character- 
istic of this type of instrument, the Faught aneroid may be 
described as follows: — • 



140 



SPHYGMOMANOMETRY AND SPHYGMOGRAPHY. 



The Faught Pocket Sphygmomanometer. — This instru- 
ment consists of a gold-plated aneroid gauge with a white- 
enameled dial, each space of which represents 2 millimeters and 
will give readings from zero up to 300 millimeters. This to- 
gether with the flexible arm-band and metal pump constitute a 




Fig. 37.— Enlarged Diagram Showing the Principle and the Working 
Parts of the Author's Pocket Indicator. 

very simple and practical sphygmomanometer, which, with its 
carrying case, can fit very easily in the coat-pocket. This in- 
strument fulfills all the demands of the blood-pressure test, 
giving accurately both the systolic and diastolic readings, from 
which can be computed the pulse-pressure and the mean. It 
has been in practical operation sufficiently long to prove not 
only that it is accurate, but that it maintains its accuracy. 



SPHYGMOMANOMETRY. 141 

making it "imnecessary to repeatedly compare the scale with the 
standard mercury column. The rubber portion of the arm-band 
measures 5x9 inches^ and so conforms with the requirements of 
the best authorities. The apparatus can be applied and removed 
in a very short time, and complete observations can be made in 
less than two minutes. It is not affected by temperature or 
atmosphere, since when the apparatus is at rest the pressure on 
both sides of the diaphragm is equal. When using this ap- 
paratus, the method is the same as directions under the mercury 
sphygmomanometer, excepting that the needle valve is placed 
upon the pump instead of on the indicator. 

The purchaser is offered the choice between two styles of 
scale: The regular pocket apparatus (see Fig. 36) and the 
clinical apparatus (Fig. 35), which possesses a 3%-inch dial 
and a scale reading to 350 millimeters, useful for hospital and 
bedside work, especially in ward-class teaching. 

The principle involved in sphygmomanometry being the 
same, regardless of the type of apparatus employed, a single 
description will suffice. 

To Operate the Sphygmomanometer. — The patient 
should be in a comfortable, easy, and relaxed position ; repeated 
and subsequent observations of the same patient should be made, 
for the purposes of comparison, under as nearly the same condi- 
tions as possible. This refers especially to arm used, and to the 
position of the patient (see below). 

The arm-band is wrapped snugly around the bared arm of 
the patient above the elbow. The tube emerging from the cuff' 
or arm-band is attached to the indicator by one nipple and the 
pump connected by a short piece of stout tubing to the other 
nipple. The escape valve, whether it be on the indicator or 
attached to the pump, is closed. 

This arrangement forms a continuous closed pneumatic 
system communicating freely with the manometer tube of the 
instrument. Now, when pressure is raised in the arm-band, by 
the pump, the amount of force exerted is shown on the dial by 
the pointer, or on- the scale by the level of the mercury; the 
readings in either case, being in millimeters of Hg, are therefore 
comparable. 



142 SPHYGMOMANOMETRY AND SPHYGMOGRAPHY. 

TO OBTAIN THE SYSTOLIC READING. 

Method by Palpation. — With one hand find the pulse at the 
wrist of the arm to which the arm-band has been applied. The 
fingers should be in a comfortable position and should under 
no circumstances be moved during the observation. Care should 
also be observed that the pulse is not cut off by undue pressure 
of the palpating fingers. 

While the pulse is thus under observation, the pressure in 
the apparatus is raised by means of the pump until the pressure 
within the constricting band is sufficient to prevent the pulse 
from reaching the wrist. Now by a fraction of a turn in the 
escape valve the pressure in the system is slowly released. Dur- 
ing this part of the procedure, a close watch should be kept upon 
the position of the needle or the height of the mercury column 
and for the return of the first pulse-beat at the wrist. The sys- 
tolic reading is made at the instant the first pulse-beat is felt 
to pass the constricting band and reach the wrist. It is ad- 
visable to repeat this procedure a few times to check the cor- 
rectness of the finding. 

AUSCULTATORY METHOD OF OBTAINING 
SYSTOLIC PRESSURE. 

As the auscultatory method of blood-pressure reading is now 
the method of choice, the reader is referred to the following 
pages, in which this technic is described in detail. Besides being 
more accurate, the auscultatory method gives definite results in 
every case. It must be remembered, however, that when this 
method is employed the systolic readings will average a few 
millimeters higher than when made by palpation. 

TO OBTAIN THE DIASTOLIC PRESSURE. 

There are four recognized methods of reading the diastolic 
pressure : — 

1. The visible. 

2. The palpatory. 

3. By means of special indicators. 

4. By auscultation. 

Experience and experimental study have conclusively demon- 
strated the first three to be unreliable. The figures obtained by 



TO OBTAIN DIASTOLIC PRESSURE. 143 

them do not always indicate the correct pressure, except possibly 
the complicated and expensive instruments of Uskoff and 
Erlanger. For this reason the discussion here will be confined to 
a consideration of the auscultatory method, first advocated about 
eleven years ago by Korotokow. 

The Auscultatory Method of Blood-pressure Beading. — 
Korotokow discovered that, by placing the bell of a small 
stethoscope over the lower end of the brachial or the beginning 
of the radial artery, a series of pulse-tones could be produced by 
the pressure of the sphygmomanometer-band in position on the 
arm above it, and that these sounds were not of cardiac origin, 
but were the direct results of the effect of compression of the 
artery by the cuff. He described, in all, three phases or sounds, 
and discovered that the first occurrence of sound over the artery 
was simultaneous with the first passage of blood under the cufi, 
and was, therefore, an accurate indication of the point of sys- 
tolic pressure. The second phase was a fair indication of heart 
strength, while the third phase was the sudden disappearance 
of all sounds and indicated the moment of diastolic pressure. 

Subsequent observers, notably Sterzing, Goodman and 
Howell, and Warfield, have described a further analysis of the 
pulse tones, and divide them into five. 

The tone phases are usually distinct and clear-cut, and 
bear a definite relation to the difference between systolic and 
diastolic pressure. For example: — 

With a normal systolic pressure of 130 millimeters and a 
diastolic of 85 millimeters the phases and their average duration 
are as follows : — 

1st phase. A loud, clear, snapping tone which immediately 
follows a silent stethoscope, and which is the index of systolic 
pressure. Duration, 14 millimeters. 

2d phase. A succession of murmurs, less distinct than the 
preceding, but yet well distinguishable. Duration, 20 milli- 
meters. This phase is dependent upon cardiac efficiency. 

3d phase. This tone resembles the first phase, but is less 
sharp and distinct. Duration, 5 millimeters. 

4th phase. A dull tone, lasting for about 6 millimeters. 

5th phase. The disappearance of all sound. 

Authorities are not yet as one on the question of the dias- 



144 SPHYGMOIVIANOMETRY AND SPHYGMOGRAPHY. 

tolic pointy older observers holding the disappearance of all 
sound to be the correct diastolic point, while later investigators, 
notably Warfield,^ hold that the beginning of the fourth phase 
is the true diastolic point. However, as the difference in read- 
ing by these two methods rarely equals more than 6 millimeters, 
the variation caused by difference of end point is but slight. 
Time alone will settle this question. 

In employing the auscultatory method, much time is saved, 
and the physician is assured of greater reliability and accuracy 
in his results, when the one operation serves to give both systolic 
and diastolic reading; at the same time conveying to the ear 
much valuable data concerning the condition of the heart and 
blood-vessels. One point, however, must be borne in mind: 
many readings in the textbooks and medical literature are based 
on the first and second methods. The auscultatory method will 
give readings of a slightly higher systolic pressure and a diastolic 
pressure of 5 to 10 millimeters lower. 

APPLICATION OF THE AUSCULTATORY METHOD. 

After arranging the apparatus in the usual manner and rais- 
ing the pressure to obliteration of the pulse, a stethoscope is 
placed over the brachial artery below the cuff. As the pressure 
is gradually allowed to fall, a pulse tone is heard as the circula- 
tion commences. This represents systolic pressure. This tone 
now undergoes a number of changes (described above) until it 
suddenly becomes very faint and almost immediately disappears. 
The reading of the" sphygmomanometer at this moment repre- 
sents the diastolic pressure. 

Cautions. — To obtain accurate and reliable clinical data 
with the sphygmomanometer, it is important that some system- 
atic technic be adhered to, and that all observations not only on 
the same patient, but in all cases, be made under as nearly the 
same conditions as possible. Attention to detail will eliminate 
largely the errors arising from such factors as position of the 
patient, presence of fatigue or mental excitement, arm used for 
observation, etc. It is also valuable to note the apparatus used, 
the time of day, the pulse rate, the sex and age of the patient. 

3 Jour. Amer. Med. Assoc, 1913, Ixi, 1254. 



PULSE-PEESSURE AND MEAN PRESSURE. 145 

Care should also be taken to see that the observation is not 
too prolonged, for the interruption of the circulation in the 
extremity will, if continued, itself cause changes in pressure. 

No Single Reading Should he Accepted When it is Possible 
to Make More than One. — It is better to see the patient a 
number of times under varying conditions before deciding what 
his average blood-pressure is. 

THE PULSE-PRESSURE AND THE MEAN PRESSURE. 

Having determined the systolic pressure and the diastolic 
pressure, the diastolic pressure is subtracted from the systolic 
pressure and the remainder is the pulse-pressure (see Fig. 33, 
page 135). 

To obtain the mean pressure, add one-half of the pulse-pres- 
sure to the diastolic pressure. 

In order to enable physicians to take the blood-pressure 
readings more accurately and to make them of greater clinical 
value to the profession as a diagnostic and therapeutic guide, 
several devices for automatically holding the stethoscope in place 
on the arm during the observation have been devised. Those 
having the stethoscope iixed and incorporated in the arm-band 
are to be condemned, because of the adventitious sounds bound 
to be produced by slipping of the compression cuff and its re- 
tainer, which, heard by the physician, are very confusing, and 
also because the conception of the idea is fault}^, as either the 
stethoscope must be on the outside of the arm, if the bag is over 
the artery, or else the bag must be interposed between the 
stethoscope and the artery, if they are both on the inner aspect 
of the arm; either arrangement preventing satisfactory work. 

The sphygmometroscope of Prendergast,^ employing a but- 
ton-like projection of the receiving disc, and a separate fabric 
band to retain the apparatus, has much to recommend it and 
in the author's hands has given great satisfaction. Even better 
than this is the bracelet stethoscope shown in working position 
on the following page. This provides an elastic metallic clamp 
for retaining purposes and by this little device the stethoscope 
can be applied and removed instantly. The button on the 

* N. Y. Med. Jour,, Jan. 11, 1913. 

10 



146 



SPHYGMOMANOMETRY AND SPHYGMOGRAPHY. 



receiving end snugly fits the elbow bend, and the '"''all metaP^ 
construction does away with all adventitious sounds. This in- 
strument is shown in Fig. 38. 

Methods of Becording Observation. — For convenience in 
study, comparison, and for future reference, it is advisable to 
formulate and adhere to some method of recording the blood- 
pressure observations. Charts which liave been found satis- 
factory for this purpose will be found on page 510. 

The Normal Blood-pressure. — Experimental study and 
clinical observation have established, within fairly well defined 




G.P.PILUNG &. SON CO. PH\LK, 

Fig. 38.— Bracelet Stethoscope in Use. 



limits, the normal blood-pressure in man, and also the extent 
of what may be termed the physiologic variation. That is, the 
extent to which the normal reading may be modified by age, sex, 
exercise, time of day, altitude, posture, etc. 

Factors which may Normally Influence Blood-pressure 
Readings. — Age: There is little difference of opinion among 
observers that 90 millimeters Hg is the minimum normal systolic 
blood-pressure in the young adult. The upper boundary is more 
difficult to establish, because it may be almost impossible to 
eliminate the possibility of pathologic influences the presence of 
which are undiscoverable. Excluding all modifying influences, 
both physical and mental, we may consider that a blood- 
pressure reading in a young healthy adult which remains con- 
stantly above 140 millimeters is abnormal and caUs for explana- 



PULSE-PRESSURE AND MEAN PRESSURE. 



147 



tion. Females usually have a slightly lower blood-pressure, as lo 
the amount of which authorities differ ; personally I believe that 
this should not exceed 10 millimeters. 

The work of H. P. Woley^ of examining 1000 healthy sub- 
jects between the ages of 15 and 60 years is a very important 

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39.— WoLEY's Chart Showing Effect of Age on Blood-pressure 
Giving Mean, High and Low Systolic Average, 



contribution to our knowledge of normal blood-pressure, the 
results of which are shown in the accompanying chart (Fig. 39) . 
Except for slight variation, the figures obtained by this obseiTer 
are in accord with the results given by our best authorities. 

8 Jour. Amer. Med. Assoc, 1910, vol. Iv, No. 2, p. 121. 



148 SPHYGMOMANOMETRY AND SPHYGMOGKAPHY. 

In children "under 2 years of age, Cook places the normal 
systolic pressure between 75 and 90 millimeters Hg. According 
to Lauder Brunton,^ the maximum pressure in children between 8 
and 14 years is 90 millimeters Hg; in youths from 15 to 21 years, 
from 100 to 120 millimeters Hg. 

As early adult life is passed we have to deal with those 
normal changes in the cardio-vascular-renal system which are the 
result of the wear and tear of every-day life, and which leave 
their mark in a lessened arterial elasticity, a lessened functional 
activity of the kidneys, and by inducing degenerative changes in 
the myocardium often eventuate in chronic myocarditis. The 
inevitable result of these changes is a gradual elevation in the 
normal systolic blood-pressure. We have now to establish new 
normals by which we may determine the pathologic. For this 
purpose I have devised and employed the following rule: Con- 
sider the normal average systolic blood-pressure at age 20 to be 
120 millimeters Hg, then for each year of life above this add one- 
half of 1 millimeter to 120. This at the age of 60 means an 
average systolic pressure of 145 millimeters Hg, which coincides 
closely with the figure given by Woley, Janeway, and others. 

Size and Temperament. — With the standard armlet the 
factor of size of the individual does not enter. Temperament, 
on the other hand, does undoubtedly affect the reading, because 
in the nervous it is often impossible to remove entirely the effect 
of psychic influence; therefore, allowance must be made for an 
abnormally high reading which will often fluctuate to a surpris- 
ing degree in a limited period of time. 

Diurnal Influence. — A record of blood-pressure taken at 
frequent intervals during twenty-four hours makes a very strik- 
ing picture. The influence of this factor is most difficult to 
estimate. Janeway suggests a variation of 60 millimeters Hg as 
the extreme. I have not seen it equal this. Authorities agree 
that the lowest blood-pressure is reached during the early hours 
of sleep and that a gradual rise occurs toward morning.'^ Dur- 
ing the day there is a physiologic rise which reaches the maxi- 
mum in the evening. 

Posture. — This should not be confounded with the effect 



6 Lancet, Oct. 17, 1898. 

7 Brush and Fairweather : Amer. Jour, Physiol,, vol. v, p. 199, 



PULSE-PRESSURE AND MEAN PRESSURE. 149 

of gravity, which may be eliminated by making all observations 
with the cuff at the level of the heart. Authorities differ ; Jane- 
way, and Erlanger and Hooker hold that the effect of posture is 
insignificant, while 0. Z. Stephens^ and A. M. Sanford^ 
demonstrate by the examination of a large number of normal 
ii:dividuals that this has a very constant and definite influence. 

Their observations, with which mine agree, show that there 
is little alteration in pressure between the standing and sitting 
postures; occasionally there is a rise of a few millimeters. Be- 
tween the standing and the recumbent Stephens reports an 
elevation often amounting to 20 millimeters Hg, while Sanford 
notes a rise amounting to only half of this. Between the stand- 
ing and the head down (Trendelenburg) the rise in pressure may 
reach 35 millimeters Hg. These observers note a compensatory 
lowering of pulse rate, upon which Schapiro has based a test for 
the functional capacity of the heart. 

Prolonged rest in bed by one accustomed to active exercise, 
especially if there was a tendency to high pressure, causes a 
rapid and marked fall with the establishment of a new systolic 
level. 

Emotion and Excitement, Including Pain. — In deter- 
mining the influence of psychic disturbances, temperament plays 
an important part. The pressure-raising effect of pain, fright, 
fear, and apprehension must always be recognized. Vasomotor 
disturbances from sensations of heat or cold and changes in the 
arm from prolonged pressure of the arm-band must not be 
ignored. Every effort should be made during the test to elimi- 
nate psychic and vasomotor disturbances by establishing a 
proper understanding between the patient and physician, and 
by completing the test with as little delay as possible. 

Exercise. — Muscular exertion in the healthy, especially if 
sharp, may cause an elevation in blood-pressure of from 15 to 35 
millimeters. This elevation becomes less marked as the subject 
becomes accustomed to performing the act or acts. This reduc- 
tion in susceptibility of the cardiovascular system is one of the 
beneficial effects of training. 

When effort is prolonged but moderate in severity (as in 

8 Jour. Amer. Med. Assoc, Oct. 1, 1904. 
9IUd,, Feb. 18, 1898. 



150 SPHYGMOJSIANOMETRY AND SPHYGMOGRAPHY. 

walking) the systolic pressure may rise from 5 to 10 millimeters, 
but soon adjusts itself to a new level upon which additional 
exertion has little if any effect, until a condition of fatigue is 
reached. Fatigue from prolonged exertion results in a fall in 
pressure, which increases until a dangerously low pressure 
is reached, if the effort is continued.!^ During moderate exercise 
in a normal healthy person the systolic and diastolic pressures 
tend to become more widely separated, i.e., the pulse-pressure 
becomes greater, i^ Upon this physiologic fact is based the 
work test of Graiipner.12 

Passive movements and massage, except when causing pres- 
sure upon the thorax or abdomen, can be prolonged without 
materially altering the systolic pressure (Eichberg). 

Diet and Digestion. — The difficulties in the way of ac- 
curately gauging the effect of diet and digestion on blood- 
pressure are great, because of the difficulty met in eliminating 
conditions bordering on the pathologic. The effect of overeating, 
overingestion of fluids, including alcohol, insufficient exercise and 
defective elimination must all be considered and weighed. 
Observers differ upon this question, some holding that as a 
result of the normal splanchnic engorgement blood-pressure falls 
a few millimeters after eating, while others affirm that observa- 
tion shows a normal rise in pressure amounting to from 10 to 20 
millimeters, and that the rise may exceed this when large quan- 
tities of fluid, particularly beer, are taken with the meal. 

Alcohol. — Clinical evidence so far shows that a moderate 
amount of alcoholic beverages taken regularly does rot ma- 
terially influence blood-pressure. The irregular use of alcohol 
occasions first a rise of from 10 to 20 millimeters follower! 
shortly by a fall as the superficial capillaries become dilated. 

Tobacco. — We find that the moderate use of tobacco in 
those accustomed to its habitual use causes at first a sedative 
action with lowering of blood-pressure, while if used immoder- 
ately the pressure is elevated. A long series of tests by the 
writer, upon three healthy subjects, showed a fall in blood- 
pressure from 4 to 10 millimeters after smoking one cigar. If 
this was followed by a second, the pressure tended to return to 

10 Karrington : Zeitschr. f. klin. Med., lOO.*]. vol. L, p. .".22. 

11 Krehl : "Clinical Pathology," 1005, 3d ed. 

12 "Die Messung. der Herzkraft," 1905. 



PULSE-PRESSURE AND MEAN PRESSURE. 151 

the original level^ while after from three to five cigars smoked 
in succession the pressure rose from 2 to 15 millimeters above 
the original reading. 

Pathologic Variations in Blood-pressure. — For convenience 
in study we may appropriately divide pathologic alterations in 
blood-pressure into pathologic high pressure and pathologic low 
pressure. 

Pathologic High Blood-pressure. — High pressure per 
se is not a disease, but a phenomenon or symptom, which may 
accompany a great variety of diseased conditions, including 
arteriosclerosis, angina pectoris (usually), acute nephritis, 
chronic interstitial nephritis, chronic parenchymatous nephritis, 
certain forms of valvular disease, acute endocarditis, chronic 
myocarditis, eclampsia, cerebral hemorrhage, arterial thrombosis, 
migraine (usually), lead poisoning, asphyxia, syphilis of heart 
and arteries. 

Pathologic Low Pressure. — A pathologic depression in 
blood-pressure may be caused by the depressing influence of cir- 
culating toxins acting either upon the heart, blood-vessels or con- 
trolling nervous mechanism or to sudden withdrawal of a large 
volume of blood from the circulation as in hemorrhage, after 
venesection, copious diaphoresis, diarrhea, or in shoch. 

The lowest blood-pressure compatible with life has been 
reported by Neu to be from 40 to 45 millimeters of mercury, and 
this only occurred with subnormal temperature in the moribund 
state. He has seen recovery after a fall in pressure down to 50 
millimeters. 

It is noted that a moderate and progressively falling pres- 
sure occurs in most progressive and prolonged fevers, as in 
typhoid fever. When due to such a cause the depression is rap- 
idly overcome and disappears as convalescence is established. 

Widespread dilatation of the vessels and consequent lower- 
ing of blood-pressure have been noted in the last stages of arterio- 
sclerosis (Krehl). 

Functional Tests in Chronic Myocarditis. — The sphyg- 
momanometer is a most valuable means of detecting alterations 
in the musculature of the heart, often before the development of 
the usual physical signs. 



152 SPHYGMOMANOMETRY AND SPHYGMOGRAPHY. 

In the physical examination the state of the superficial 
vessels, together with the pulse-rate and particularly the reaction 
of the heart to posture and exercise as determined by the 
sphygmomanometer, is all-important. This latter may be deter- 
mined by the following tests : — 

Work: Test. — Moderate exertion raises pressure in normal 
hearts and this rise is sustained during it if not unduly severe 
or prolonged. In weakened heart muscle from any cause a 
primary rise may occur, but is quickly followed by a fall ; in the 
most serious a fall occurs from the first. 

Graupnee^s Test. 12 — This is based upon the physiologic 
fact that a given amount of exercise, such as ten bending move- 
ments or running up a flight of stairs, causes an acceleration in 
the pulse-rate and an elevation in blood-pressure. But the latter 
does not appear coincidently with the former; or, if, as in some 
cases, the pressure does rise first, it fails to rise again after the 
pulse has returned to normal. It is this secondary rise which 
indicated a good heart muscle. A not too seriously affected 
heart may show a rise in blood-pressure immediately after the 
exertion, but with the slowing of the pulse the pressure will be 
found to have fallen to a level lower than before the experiment. 
The sphygmomanometer is required for an accurate demon- 
stration of these changes in pressure, which may be recorded in 
definite units of measure for future reference and comparison. 

ScHAPiRO^s Test. — This is based upon the alteration in 
pulse-rate occurring in normal individuals on change of posture 
from the standing to the recumbent. Normally, the number of 
pulse beats per minute is from seven to ten less in the recum- 
bent position; but when chronic myocarditis develops this dif- 
ference tends to disappear, so that in seriously weakened hearts 
the pulse may be as rapid in the recumbent as in the sitting 
posture. 

Cautions. — It is not advisable to apply this test to patients 
with excessively high llood-pressure, in those of apoplectic tend- 
ency, or in those with high-grade arteriosclerosis. The test is 
unsafe in those with a systolic pressure of 200 millimeters or 
over. In such cases tnere is danger of ocular or cerebral hemor- 
rhage or acute dilatation of heart. 

J3 Berlin. cli». Wochen., xliy, 1907, Nq. 15- 



PULSE-PRESSUEE AND MEAN PRESSURE. 153 

The test will be difficult if not impossible of application in 
women unless all tight clothing is removed. 

Valvular disease is not necessarily a contraindication to this 
test, as the condition of the myocardium seems to be the only 
important factor, except in aortic regurgitation with high pres- 
sure, so that the presence of valvular lesions need not detract 
from the value of the information obtained by this test. 

In life-insurance examinations 1 4 it is now well recognized 
that such pathologic changes may be present in the cardiovascular 
and renal systems long before any suggestive symptoms are com- 
plained of by the individual, or if any complaint is made it is 
usually attributed to some trivial cause. 

In the presence of such a beginning arteriosclerosis the 
blood-pressure need not be greatly increased; an elevation of 30 
to 40 millimeters above that estimated as normal for the in- 
dividual is significant and demands explanation. On the other 
hand, a rise of even this amount should never be hastily assigned 
to arteriosclerosis and the risk therefore rejected without further 
study. 

It is necessary to recognize in this connection the activity 
of other and less important factors, such as the alimentary 
hypertension, so well described by Eussell, occurring in normal 
vessels due to errors in diet of either quantitative or qualitative 
origin and responding immediately to the correction of such 
errors, together with stimulation of the eliminative functions. 
Of further interest, particularly to the life-insurance examiner, 
are the so-called physiologic variations, caused by age, sex, 
mental and physical excitement, fatigue, etc. These must all 
be taken into consideration in estimating the character and class 
of the risk. (See page 146.) 

Such variations need not confuse the examiner, as they 
all occur within a range sufficiently restricted to prevent them 
from obscuring the issue. The only one which needs special 
consideration is the age factor. To determine this, employ the 
author^s formula explained on page 148. 

High Pressure and Transient Albuminuria. — Probably the 
most confusing combination of symptoms met is the case 

14 "The Insurance Examiner and the Blood-pressure Test," F. A. Faught, 
Medical Record, Aug. XO, 1912. 



154 SPHYGMOMANOMETRY AND SPHYGMOGRAPHY. 

which presents a slight hypertension and an occasional trace of 
albumin in the urine. These cases are best examined at tlie 
home or branch office and should be referred there whenever 
possible, for it is only after very careful and complete examina- 
tion with repeated urine and blood-pressure tests that a correct 
conclusion regarding the safety of the applicant can be reached. 
If after eliminating the possibility of an alimentary hyper- 
tension a distinct elevation in pressure remains with albumin in 
the urine, even in occasional minute traces, the risk is doubt- 
fully good, while if accomplished by accentuation of the aortic 
second sound or casts the risk is bad and calls for rejection. 

B. SPHYGMOGRAPHY. 

Sphygmography is the method of registering the pulse wave 
occurring in some peripheral vessel, generally the radial artery, 
upon a moving surface (usually smoked paper) by means of a 

special instnmient, the sphygmograph. 

The apparatuses which are employed for this study are, even 
the simplest of them, complicated and difficult to manage. The 
essence of the method should be simplicity, for the more com- 
plicated the procedure, the more unsuitable it becomes for prac- 
tical clinical purposes. This requirement does not obtain in 
hospitals with large staffs and much clinical assistance. 

The Sphygmograph. — It is scarcely necessary to enter into a 
full account of the construction of the various sphygmographs. 
They are practically all constructed on the same principle. A 
steel spring is laid upon the radial artery at the wrist in such 
a manner that, while it compresses the artery, it does not ob- 
literate it. Attached directly to the spring is a long lever, or a 
series of small levers, that magnify the movements of the spring. 
The free extremity of the lever presses lightly against a strip 
(jf paper whose surface has been blackened by the smoke of burn- 
ing camphor or turpentine, the strip of paper passing at a 
uniform speed by means of a clockwork arrangement. The 
>implest and most practical sph3^gmograph is that of Dudgeon 
(Fig. 40), which, after a little practice, may be relied upon to 
give a true and accurate record of the pulse wave, 



SPHYGMOGRAPHY. 155 

The Polygraph (Fig. 41). — There are many perceptible 
movements due to the circulation that the sphygmograph fails 
to register, and when it is desired to record these movements 
other instruments have to be used. The method most commonly 
employed has been to convey, by means of a tubular air system, 
the movements to be registered, to a recording tambour operating 




Fig. 40.— Dudgeon's Sphygmograph. 



against a suitable recording surface. Two tambours are usually 
employed, with their levers placed one above the other, so that 
simultaneous records of these different movements, the apex beat 
and jugular pulse, can be readily reproduced. 

Jaquet's polygraph consists of a metal frame to which is 
secured a cuff for attaching to the wrist. The sphygmograph 
proper is attached to the frame. The window cut out of the 
frame is to be applied accurately along the radial artery (pre- 
viously marked out with a pencil), and the cuff then strapped 
around the wrist quite tightly. The sphygmograph proper is set 



156 



SPHYGMOMANOMETRY AND SPHYGMOGRAPHY. 



into the frame by hooking into the hinge, and then the connec- 
tion of the two parts is effected by pressing down at and tighten- 
ing the screw. The pulse-registering apparatus consists of a 
short, broad spring, which presses upon the artery and transmits 
its movements to the registering-needle by means of the lever 
system. The screw also serves to adjust the registering-needle 
at the desired height upon the smoked strip of paper. By screw- 




FIG. 41.— JAQUET POLYGRAFH. 

ing it down, the spring is pressed against the artery. The screw 
is connected with an "eccentric" contrived to increase or diminish 
the pressure upon the spring. The amount of pressure can be 
determined by noting the position of the figures upon the screw, 
With this mechanism the instrument can be adjusted with prac- 
tically equal pressure in each case, and taken away from the 
frame and reapplied in the same case, without alteration of the 
pressure. The paper is run through in a horizontal position and 
it may be made of any convenient length. A little box contains 
the clockwork which moves the strip of paper, which is con- 
trolled by pressing down on the lever. The rate that the paper 



NORMAL PULSE CURVE. 157 

moves may be increased from 1 to 4 centimeters in a second 
by altering the position of the lever. The slower motion pro- 
duces a curve which presents a better general idea; the more 
rapid motion, one which may be more accurately analyzed. This 
rate motion may be altered while the sphygmograph is in action. 
The box also contains a .watch actuating a time-registering 
mechanism. The latter consists of a small stylus which registers 
a mark upon the margin of the smoked ribbon every one-fifth 
second. 

The Ink Poly^aph. — This simple and useful device has 
been perfected by MacKenzie; it records in ink on a roll of 
paper, which can be of any desired length, at the same time 
obviating the inconvenience of blackening and varnishing the 
paper. In operation this instrument is very similar to the one 
just described. 

EXPLANATION OF A NORMAL PULSE CURVE; FACTORS 
WHICH INFLUENCE ITS FORM. 

The curves obtained with the good modern sphygmographs 
usually correspond quite uniformly. The pulse wave is com- 
posed of a steep ascending and a rather slanting descending 
limb, in which the sphygmographic tracings show a number of 
small elevations in the descending limb (so-called catacrotic ele- 
vations). Similar irregularities which may appear under path- 
ologic conditions in the ascending limb are termed anacrotic 
elevations. A pulse with catacrotic elevations is termed cata- 
crotic; one with anacrotic elevations, anacrotic. 

Generally speaking, the curve varies under the following 
circumstances (MacKenzie) : — 

1. Other things being equal, the curve is lower the higher 
the mean blood-pressure, and vice versa. This can be under- 
stood easily, because with a high blood-pressure the arterial wall 
is already so tense that even during diastole the increase in 
pressure during systole can produce but a very small excursion. 
When, on the contrary, the pressure is low, the artery easily 
yields to the change. Of course, this does not apply to those 
cases where the high pressure is produced by a large systole (left- 
sided cardiac dilatation and hypertrophy in compensated heart 



158 SPHYGMOMANOMETRY AND SPHYGMOGRAPHY. 

affections), nor where a low pressure is due to a small systole 
(disturbances of compensation). 

2. Other things being equal (the same blood-pressure), the 
pulse curve is higher the larger the systole, and vice versa. 

3. Other things being equal (equal systole and equal blood- 
pressure), the ascending limb of the curve is steeper the quicker 
systole takes place. 

4. Other things being equal, a low mean blood-pressure 
produces both a steep ascent and a steep descent, i.e., a pointed 
curve (celerity of curve in toto). Conversely, a high blood- 
pressure produces a slanting rise and a gradual descent (tardiness 
of curve). 

5. Other things being equal, rigid arterial walls (like high 
blood-pressure) produce low curves with slanting ascent and 
gradual descent (tardiness). On the contrary, delicate elastic 
arteries (like low blood-pressure) produce curves with steep 
ascents and descents (celerity). 



VII. 

ANIMAL PARASITES 



PARASITES IN THE BLOOD. 

THE PLASMODIUM OF MALARIA. 

This is the only sporozoa found in the blood which is con- 
nected with disease in man, although numerous hemosporidia 
have been reported in many of the lower animals. 

History of the Malarial Parasite. — This is one of the most 
interesting chapters in medicine. The parasite was discovered 
by Laveran in 1880, but it was not until 1885 that Golgi observed 
that sporulation occurred simultaneously with the malarial 
paroxysm. Golgi also demonstrated the existence of different 
species for different types of fever. 

Life History. — When man is first infected there commences 
a non-sexual cycle which is completed in forty-eight or seventy- 
two hours, depending upon the species of parasite. The 
sporozoite bores into a red cell, assumes a spherical form, and 
continues to enlarge. As it approaches maturity, it shows signs 
of division into a varying number of spore-like bodies. The para- 
site at this stage is termed a merocyte. When the merocyte 
ruptures, these spore-like bodies, or sporozoites, each enter a fresh 
red cell and develop as before. At the time these merocytes 
rupture, it is believed that a toxin is liberated which causes the 
malarial paroxysm. The cycle goes on by geometric progression. 
From the first indication of the sporozoite it is usually two weeks 
before a sufficient number of merocytes rupture simultaneously 
to produce toxic symptoms (the period of incubation). This 
cycle is termed schizogony. After a varying time sexual forms 
develop. These are termed gametes, and show two types, one 
which contains more pigment, has little chromatin, and stains 
more deeply; this is the female, or macrogameU; in the other 
there is little pigment, much more chromatin, and it stains less 
deeply; this is the male, or microgametocyU. 

(159) 



160 ANIMAL PARASITES. 

When the gametes are taken into the stomach of the 
mosquito (Anophelinas), the male cell shows tail-like projections 
which have an active lashing movement, which break off from the 
cell carrier and are thereafter termed mio^o gametes. These 
enter the macrogametes and the combination forms a zygote. 
The zygote enters the epithelial layer of the stomach of the 
mosquito, where it continues to enlarge for about one week. 
When it has reached the size of about 60 microns it is seen to 
contain hundreds of delicate falciform bodies. The mature 
zygote now ruptures, and the sporozoites are thrown off into the 
midgut of the mosquito, whence they make their way to the 
salivary glands, from which they are introduced into the circula- 
tion of the person bitten by the mosquito, and then start the 
non-sexual cycle already described. As the sexual cycle takes 
place into the mosquito, this insect is the definitive host, while 
man is the intermediary host. 

There are three species of malarial parasites : first, P. vivax, 
or benign tertian, with a cycle of forty-eight hours; second, the 
P. malarice, or quartan, with a cycle of seventy-two hours, and, 
third, the P. falciform, or estivo-autumual or malignant tertian, 
with a cycle of forty-eight hours. 

Variations in cycle may be produced by infections from 
mosquitoes on successive nights, so that one will mature and 
sporulate twenty-four hours before the second. This will give a 
quotidian type of fever. In the estivo-autumnal infections ac- 
celeration or retardation in sporulation will cause a very pro- 
tracted paroxysm lasting from eighteen to thirty-six hours. 
This tends to give a continued fever instead of the characteristic 
type. 

In the diagnosis of malaria one should also examine both 
the fresh and the stained specimen, as each gives valuable in- 
formation in differentiating species. When time will not per- 
mit the examination by both methods, always use the smear 
stained by Leishmann^s stain, as the small, externally situated 
rings of estivo-autumnal fever may escape notice in a fresh 
specimen. 

It is evident that, while there is considerable similarity 
among the three varieties of parasites, there are, nevertheless, 



DESCRIPTION OF PLATE V. 

Malarial Parasites, (Kolle & Wassermann.) 

1. Two tertian parasites about thirty-six hours old, attacked blood- 
corpuscles magnified. 

2. Tertian parasite about thirty-six hours old; stained by Roman- 
owsky's method. The black granule in the parasite is not pigment but 
chromatin. Next to it and to the left is a large lymphocyte, and under 
it the black spot is a blood-plate. 

3. Tertian parasite, division form nearby is a polynuclear leukocyte. 

4. Quartan parasite, ribbon form. 

5. Quartan parasite, undergoing division. 

6. Tropical fever parasite (^stivo-autumnal.). In one blood- 
corpuscle may be seen a smaller, medium, and large tropical fever-ring 
parasite. 

7. Tropical fever parasite. Gametes half-moon spherical form. 
Smear from bone marrow. 

8. Tropical fever parasite, which is preparing for division heaped up 
in the blood capillaries of the brain. 

Asexual Forms 

9. Smaller tertian ring about twelve hours old. 

10. Tertian parasite about thirty-six hours old, so-called ameboid 
form. 

11. Tertian parasite still showing ring fever, forty-two hours old. 

12. Tertian parasite, two hours before febrile attack. The pigment is 
beginning to arrange itself in streaks or lines. 

13. Tertian parasite further advanced in division. Pigment collected 
in large quantities. 

14. Further advanced in the division. (Tertian parasite.) 



PLATE V. 



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E 


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[ *i ^ 1 

1 T' M 


3 




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3 



DETECTION OF PLASMODIUM. 



ir,i 



certain peculiarities and characteristics which enable the export 
to quite positively differentiate between them. 



DIFFERENTIAL DIAGNOSIS OF PLASMODIA. 



TERTIAN. 

Develops in 48 hours. 

Pale and indistinct. 

Actively ameboid. 
Pigment fine. 

Pigment actively mo- 
tile. 
Pigment light. 
Full size of corpuscle. 

Degeneration forms 
twice the size of 
corpuscle. 

Segments 16 to 20. 

Irregular segments 
are frequent. 

Corpuscles large, 
colorless, and swol- 
len. 



QUARTAN. 

Develops in 72 hours. 

Sharp outline; re- 

fractile. 
Slightly ameboid. 
Pigment coarse. 

Pigment slow in move- 
ment. 

Pigment dark. 

Smaller than corpus- 
cle. 

Degeneration forms 
vary; much smaller 
than tertian. 

Segments 5 to 12. 

Beautiful rosettes. 

Corpuscles shrunken 
and brassy. 



EGTI VO- AU TU MNAI . 

Develops in 24 to 48 

hours. 
Has irregular appear 

ance. 
Actively ameboid. 
Pigment granules very 

few. 
Pigment still. 

Pigment light. 
Smaller than corpus- 
cle. 
Absent. 



Segmentation forms 
not seen in periph- 
eral blood. 

Corpuscles shriveled, 
brassy, not decolor- 
ized. 

Forms crescents. 



THE DETECTION OF THE PLASMODIUM. 

In searching for the organism it is always desirable, when 
possible, to examine the fresh specimen at the bedside of the 
patient. If this is impracticable, warm the slide and seal the 
cover with a little vaselin, and the organisms will retain their 
motility for at least a couple of hours. 

If considerable time must elapse before the examination, 
films should be made and dried preparatory to staining, which 
can then be done at a convenient time. 

Staining Methods. — Do not have the blood-films too thick; 
the individual cells should not overlap; there should be no 
rouleau formation. Allow the films to air-dry without heat and 
then, without previous fixing, stain with one of the polychrome 
stains. Wright's is very satisfactory (see Appendix for stain). 
Do not give up the search until all the films have been examined 
thoroughly. A number of fields may show no organisms; then 
some may appear. The specimen is best obtained either eight or 
ten hours before or after a chill, as at this time the organisms 

11 



163 ANIMAL PARASITES. 

are most likely to be in the peripheral circulation. Medication 
should be withheld for as long a time before the specimen is 
taken as possible. 

What to Examine For. — Fresh specimen: Look for red 
cells containing actively moving black specks (pigment granules 
and living protoplasm). Unusually pale cells containing clear 
areas which are irregular and constantly changing shape. Extra 
large red blood-cells. 

After a little experience the pigmented organisms are read- 
ily distinguished. Violent commotion among a group of red 
cells will direct attention to the flagellate. The hyaline bodies 
are usually the most difficult to identify without experience. 
The apparent, but not real (artefact), may be found in large 
numbers; these are usually distorted and deformed cells that 
have become altered in the preparation of the specimen. Finally, 
pigment-bearing leukocytes may be found in excess in malaria. 
These are, as a rule, polymorphonuclears which have become 
phagocytic, and have taken up the iron pigment set free by the 
malarial organisms. 

Examination of the Stained Specimen. — Unless one has had 
some experience, the stained specimen will yield much more 
definite results than the fresh, provided that the staining technic 
is good. The slight refractility, both of the cell and of the 
parasite, in the fresh specimen makes it difficult to get the 
proper illumination. In the hands of an expert the examination 
of fresh blood is usually all that is required for a diagnosis in 
the average case, and when the parasites are moderately 
numerous even the beginner can scarcely make a mistake. It 
would seem, therefore, inadvisable to settle the diagnosis after 
an examination of the fresh specimen in case no organisms are 
found, but to control this examination by a careful study of a 
stained specimen, where one may frequently be surprised at the 
number of parasites seen. 

Caution. — Frequently one observes in stained specimens 
many artefacts due to deposition of staining pigments upon the 
red cell (see above), while in the fresh specimen areas of co- 
agulation necrosis are not infrequently seen, so that the beginner 
may incorrectly assume the presence of malarial organisms. For 
an absolute diagnosis of malaria, it is necessary to find intracel- 



PARASITE OF SCARLET FEVER. 153 

lular organisms, and not to be content with a single examination 
in doubtful cases. 

CULTIVATION OF MALARIA PLASMODIA. 

C. C. Bassi on several occasions has reported his work in 
the cultivation of the three species of the malarial parasite. In 
order to cultivate the malarial parasite, Bass recommends media 
containing human serum, Locke's fluid (from which the cal- 
cium chlorid is omitted), and human ascitic fluid. The addi- 
tion of a percentage of dextrose facilitates the growth. The sus- 
pected blood is transferred to this medium, in which it is found 
that the plasmodia grow in a thin layer near the top of the 
cell sediment, beneath which zone the parasites all die. Para- 
sites develop readily in the red corpuscles, but are only main- 
tained alive while in the human red blood-cells, as they are de- 
stroyed by the leukocytes, by serum, Locke's fluid, ascitic fluid, 
etc., as soon as liberation occurs. 

The most favorable temperature for cultivation is about 
40° C. The latest report of this observer shows positive cultures 
obtained in 29 cases of the estivo-autumnal type, in 6 cases of 
tertian, and in 1 case of quartan. Cultures have been carried out 
as far as four generations. Only the asexual cycle has been 
observed. The chief advantage to be derived from the employ- 
ment of this method would seem to be the fact that in the 
proper surroundings a small ring body will rapidly be developed 
m the segmentation form, thereby rendering microscopic recog- 
nition of this infection less difficult. 

THE PRESENT STATUS OF THE PARASITE OF 
SCARLET FEVER. 

Owing to the obscure origin, frequency, and seriousness of 
complications, many observers have directed their special efforts 
toward the finding of the specific cause of this disease. It has 
been more or less generally accepted that one or more of the 
strains of streptococcus were closely involved in the production of 
the disease and also are important determining factors in the de- 
velopment of many of the complications. Recently Klimenko^ 

1 Journal A, M. A., Part 1, September 21, 1912. 

2 Centralblatt fUr Bakter., Bd. 65, p. 45. 



164 ANIMAL PARASITES. 

published a series of exhaustive bacteriologic studies upon 523 
cases of scarlet fever in an effort to determine this relation. His 
results are important. In only 11 cases was a streptococcus re- 
covered from the blood during life^ yet these cases were all of a 
complicated or serious variety. The organisms, according to this 
observer, seem to have no relation to kidney, heart, or joint 
complications even when present. From his studies he con- 
cludes that the streptococcus is never present in the blood in the 
early stage of scarlet fever, and that it bears no causal relation 
either to the disease or to its complications. We must, there- 
fore, consider the probability that the streptococcus bears no 
direct relation to scarlet fever. 

Bohle^ reports finding certain bodies in the polymorpho- 
nuclears in scarlet fever. These he calls ^^leukocytic inclusion 
bodies.^' They are round, oval, or rod-shaped, and vary greatly 
in size. They stain readily with all the usual blood stains and 
are basophilic. They do not show any chromatin staining. He 
believes these to be of certain diagnostic value, as in one series 
they were found regularly in the blood of 30 patients with scarlet 
fever, but rarely in other conditions. 

His observations have been in part confirmed by others. 

The same observer has later described a spirochsete occur- 
ring in the leucocytes in scarlet fever, and that the "in- 
clusion bodies" may represent portions of these, which may in 
turn be the cause of disease. These observations yet re- 
main to be confirmed. 

Dr. John Kilmer^ states that he found these "inclusion 
bodies" in 94 per cent, of 49 cases of scarlet fever, during the 
first three days, but that they were rarely present after the ninth 
day. They were also found in the same percentage of cases of 
diphtheria. If these observations are confirmed, we must con- 
clude that the diagnostic value of these bodies is limited. In 
serum sickness with a scarlatiniform rash their absence excludes 
scarlet fever, while their presence in this condition may not 
necessarily mean scarlet fever. They have, therefore, a more 
negative than positive value. The technic of examination is 
extremely simple, requires very little time, and should be em- 
ployed as an aid to differential diagnosis. 

3 Centralblatt fiir Bakter., Bd. 61, p. 63. 

4 American Journal of Diseases of Children, vol. iv. 



FILARIASIS. 165 

Blood-smears are made in the usual way, stained by one of 
the accepted methods, and examined for the presence of baso- 
philic bodies of varying size, which are round, oval, or rod-shaped 
and found in the polymorphonuclears. 

THE PARASITE OF YELLOW FEVER. 

Aristides Argamonte,^ doubts that the so-called Seidelin 
bodies are the cause of yellow fever. He states that defects in 
staining technic may produce similar bodies, which are probably 
nuclear and protoplasmic fragments. However, it is by no 
means settled that these bodies do not represent some stage in 
the development of a parasite which is the cause of yellow fever. 
They may be demonstrated in an ordinary blood-smear taken 
under ordinary precautions •from a patient suffering with yellow 
fever, and are stained best by one of the differential stains, of 
which Seidelin recommends the Giemsa reagent. 

H. Noguchi recently reports having isolated and cultured a 
leptospira which he claims to' be the cause of yellow fever. This 
he has termed the Leptospira ideroides. 

FILARIASIS. 

This is a condition associated with the presence of filariaj 
in the blood (Filaria sanguinis h o minis) . While many of these 
filarige are known, the most common one is the Filaria hancrofti 
(Filaria nocturna) (see also page 190). For the special dis- 
tinguishing characteristics larger works must be consulted. 

The adult or parent organism is slender and thread-like, 
varying from 3 to 6 inches in length. It inhabits the lymphatics 
and tissues, while the embryos appear in the peripheral 
circulation. 

Originally these embryos were supposed only to appear in 
the circulation toward evening, their numbers gradually rising to 
a maximum at about midnight and diminishing toward dawn. 
This rule has been found by Eivas and Smith <5 to have excep- 
tions, as shown by findings of F. nocturna and F. diurna in the 
same specimen of blood. During the day they are found in the 
internal organs, especially the lungs. The forms appearing in 

5 N. Y. Med. Record, August 17, 1912, p. 288. 

6 D. Rivas and Allen J. Smith, So. Med. Jour., p. 631. 1913. 



166 ANIMAL PARASITES. 

the blood are practically all embryos, as the adult type lies in the 
lymphatics, where it may obstruct the lymph flow. The move- 
ments of these embryos is at first distinctly progressive, as seen 
under the microscope; but they soon become motionless, appear- 
ing to attach themselves to the glass slide at their anterior end. 

The obstruction in the lymph-glands may also be brought 
about by the eggs, which are 25 to 38 microns long by 15 broad. 
The embryos reach the general circulation only through the 
thoracic duct. 

Like the malarial organisms, the filaria has an intermediate 
host in the mosquito, both of the culex and anopheles. The 
embryos, which are taken up by the bite of the mosquito, cast 
off their sheath in about one hour in the stomach of a mosquito. 
Some of these embryos die at this stage, but others bore actively 
through the intestinal wall, to the muscle, where they remain. 
During the next two or three days, the embryo becomes larger 
and its alimentary tract develops. On the seventh day the worm 
is about 1% millimeters long and is perfectly formed. It now 
travels toward the head and takes its position in the labium, 
whence it enters the blood of its new host during the biting of 
the insect. A large number of these adult forms is necessary 
to cause very severe cases and many years may pass before any 
symptoms are manifest. 

In examining the blood for filaria, it is best to take a speci- 
men late at night and to make a very thick smear, which should 
be examined fresh with a low power. 

Besides the ordinary anemia which may develop in such 
cases, we find a very striking eosinophilia, which may run from 
5 to 20 per cent. 

A very characteristic finding in such cases is the condition of 
hematoehyluria followed by chyluria. This hematochyluria 
seems to be due to rupture of the varicose lymph-vessels of the 
bladder, which form a large part of the collateral circulation 
when the thoracic duct is occluded. Such attacks may occur for 
years and be separated by long intervals. Their onset is spon- 
taneous or follows exertion and is usually associated with pain 
and fever. The urine shows the presence of blood, chyle (as 
high as 3.8 per cent, fat), and embryos (Emerson). 



SLEEPING SICKNESS. 167 

Many other forms of filarise are known, but this Bancroft 
type seems to be the most important. While this disease ocenrs 
endemically in the tropics, it is advisable in a case showing lymph 
tumor, elephantiasis, and hematochylnria, especially when pain 
and fever and enlarged spleen are present, to examine the blood 
for the Filaria hancrofti. . 

METHOD OF EXAMINATION FOR FILARIA. 

Eivas and Smith''' recommend the employment of large 
amounts of blood especially if drawn during the day; otherwise 
the embryos will not be found. An experimental study showed 
that under the same conditions the examinations of dried and 
stained specimens greatly increased the percentage of successful 
examinations. By any method, much depends upon the time of 
drawing the blood. 

Acetic Acid Method. — Collect 0.1 to 1 cubic centimeter of 
blood from the finger in 5 to 10 cubic centimeters of a 2 per 
cent, acetic acid solution. After shaking and centrifuging, fresh 
slides are made from the sediment, and examined under the 
microscope. This method gives positive findings in most cases 
of filariasis, regardless of the hour at which the blood is collected. 

Dry Stained Method. — Spread and dry a thick smear of 
blood. Fix in any convenient way. Hemolize with distilled 
water or 2 per cent, acetic acid, wash and stain with hematoxylin 
and eosin (for stains, see Appendix). 

SLEEPING SICKNESS OR TRYPANOSOMIASIS. 

This disease is very prevalent in Central and West Africa, 
and is due to the presence of an actively motile, fusiform flagel- 
late known as the Trypanosoma gamhiense, which can be found 
in the blood-plasma (never intracorpuscularly) . It moves with 
a screw-like motion among the red cells, which it does not seem 
to disturb. This parasite, doubtless, has a sexual development. 
Kline has demonstrated that the Olossina palpaUs is the true 
host of the Trypanosoma gamhiense, although other insects may 
mechanically transmit it.^ 

7 Loc. cit. 

8 Gould and Pyle, 1912. 



168 ANIIVIAL PARASITES. 

T. Gambiense. — Castellani gives its dimensions as from 16 
to 24 microns long, and from 2 to 5 microns broad. Anteriorly 
it is either pointed or rounded and along one border is an un- 
dulating membrane, which is a thickening of the ectoplasm. 
This is continued posteriorly into a free flagellum (see C, Plate 
VI). The origin of the undulating membrane takes place from 
a minute spot of nuclear material of oval shape, called the kineto- 
nucleus, situated near the anterior end of the trypanosome. 
About the middle of the body of the parasite is an oval mass of 
nuclear material, irregularly shaped, called the trophonucleus. 
A few chromatin granules may be seen posterior to the tropho- 
nucleus, and the cytoplasm is continued in a narrow, 
diminishing band extending for some distance along the flagel- 
lum. In Castellani's description, in the "Eeports of the Eoyal 
Society on Sleeping Sickness," are pictures showing, besides the 
ordinary forms, multinucleate, polyflagellate, and non-flagellate 
forms. 

Occurrence of Parasites in Man. — The majority of observers 
believe that the parasite is found in the peripheral circulation 
and lymph-glands during the early stages (the so-called trypan- 
osomatic fever), and that the invasion of the cerebrospinal fluid 
marks the beginning of the later stages. 

These parasites vary much in number, sometimes being ab- 
sent from the peripheral circulation for a long period and then 
suddenly reappearing in large numbers. Symptoms of the dis- 
ease seem to bear but little relation to the number of parasites 
in the peripheral blood, so that in some cases it may be necessary 
to examine the fluid in the edematous areas or even to puncture 
the cervical lymph-glands. 

Methods of Isolating. — ^Various methods have been devised 
for demonstrating the tr3rpanosome in the blood of man. Unless 
the trypanosomes are quite numerous they may be overlooked in 
the ordinary fresh blood preparations. The use of a large hang- 
ing drop, and a search of from ten to fifteen minutes, will give 
positive results in a larger percentage of cases. After mounting, 
the trypanosome should be located first with the low power of 
the microscope by the movements of the red blood-cells as they 
are lashed by the flagellum of the parasite. Further investiga- 
tion should then be made with the high power. The surest 



RELAPSING FEVER. 169 

method of demonstrating this organism in fresh preparations is 
by the use of the centrifuge. About 10 cubic centimeters of 
blood or cerebrospinal fluid are drawn in the usual manner into 
a 1 per cent, sodium citrate solution or into normal salt solution, 
to prevent coagulation, centrifuged and diluted three times, and 
the third or fourth residue examined. 

Methods of Staining. — For staining blood preparations the 
various modifications of the Eomanowsky stains give very good 
results. Koch recommends the use of a heavy drop of blood, 
stained with a dilute solution of G-iemsa's stain. In the hands 
of experts this is a very reliable method, but one or two trypan- 
osomes may be overlooked on account of their being concealed by 
red blood-cells and fibrin. 

When these parasites are stained with a polychrome dye they 
show a rather large nucleus about the middle, a centrosome stain- 
ing intensely in a vacuole-like area near the blunt posterior end, 
and a line of chromatin, making a dense red stain, running down 
the edge of the undulating membrane, and terminating in the 
flagellum, which is also stained red. The protoplasm of the 
body takes a distinct blue stain. 

The parasite contains no pigment and, therefore, obtains its 
nourishment from the plasma and not from the red cell. 

In appearance Glossina palpalis is a dark-colored fly, about 
8 to 12 millimeters long. A point of recognition is the arrange- 
ment of the fly's wings in the resting position. They overlap 
like a pair of scissors. This point differentiates the tsetse fly 
from the other blood-sucking diptera with which it is associated. 

There are many other types of trypanosomata, but the 
gambiense form is the more important. This is pathogenic 
for man, but cannot be distinguished from the trypanosoma 
of the tsetse fly, which is so fatal to the horse and mule ( Trypan- 
osoma Brucei), that of the surra disease {Trypanosoma Evansi), 
or that of dourine {Trypanosoma equiperdum) . 

RELAPSING FEVER (FEBRIS RECURRENS; SPIRILLUM 
FEVER; FAMINE FEVER; SEVEN-DAY FEVER; TY- 
PHUS ICTEROIDES). 

The Organism. — ^The specific cause of relapsing fever is the 
spirochseta (spirillum) Oberaieieri, formerly regarded as a bac- 



170 ANIMAL PARASITES. 

terium of the genus spirochaeta, but now regarded as probably a 
protozoan parasite — a trypanosome. First discovered by Ober- 
meier in the blood of persons ill with the disease, it is known by 
his name. It is a narrow spiral about 0.025 to 0.05 millimeters 
(16 to 40 microns) in length; that is, its length is from 3 to 6 
times the width of a red blood disc and about 1 micron in width. 
It is thin, sharply curved, and appears to be structureless. 

The Fresh Specimen. — Blood may be prepared after the 
same manner as that employed in searching for the malarial 
parasite and should first be examined by the medium power in 
order to more easily locate the parasites. 

It is seen in the blood only during the febrile period of the 
disease, and at that time is actively motile, with a rapid, wavy 
motion, much resembling the movements of a coiled spring in its 
stretching and collapsing. It moves rather slowly among the 
corpuscles, but does not disturb them to any extent. Before the 
crisis and in the intervals the organism is not found; but small, 
glistening spherules, said to be its spores, take its place. Con- 
firmation of the contagious nature of the disease is found in the 
fact that it has been communicated from one human being to 
another by inoculation of blood, and to monkeys in the same way. 

Staining Method. — Smears of blood taken at the proper time 
(during the febrile period) are made and dried in the usual man- 
ner, after which they should be stained by one of the simple 
stains. 

The organism takes a deep chromatin stain and also stains 
with methylene-blue in from two to five minutes. 

Loewenthal has applied the agglutination test to the blood of 
suspected cases and found the reaction positive in 85 per cent, of 
the cases during the periods in which the parasites are absent. 

Pathologic Change. — There is no essential morbid anatomy, 
and such as is found corresponds with that of typhus. Most con- 
spicuous is enlargement of the spleen. 

The changes in the blood are not characteristic of this con- 
dition. The red blood-cells seem to be slightly diminished in 
number for several days after the beginning of the attack, but 
rapidly return to normal during the afebrile period. The hemo- 
globin may be reduced to as low as 50 per cent., so that a very 
distinct anemia of the secondary type is common. The leuko- 



KALA-AZAR. 171 

cytes seem to be distinctly increased in this disease, the most 
marked leukocytosis occurring just after the crisis, which sub- 
sides to, or nearly to, normal, during the afebrile period of the 
disease. 

KALA-AZAR (TROPICAL SPLENOMEGALY; CACHEXIAL 
FEVER; DUMDUM FEVER). 

Through the researches; of Donovan, Leishmann, and Eoss, 
parasites have been demonstrated in the blood which are prob- 
ably directly associated with the condition known as kala-azar. 
The organism is usually known as the Leishman-Donovan body, 
and is a small, oval, round, or oat-shaped body from 2 to 3 
microns in diameter. These bodies have a definite cell outline 
and contain two chromatin masses. The larger one, or nucleus, 
is almost round or oval and stains faintly, while the smaller, 
bacillus-shaped centrosome stains deeply and is directed almost 
at right angles to the axis of the nucleus. These two chromatin 
masses are both in the long axis of the cell, and the outline of 
the cell cannot always be seen, although these two masses thus 
arranged are distinctive. These bodies probably represent a stage 
in the development of a trypanosome, as shown by the work of 
Leishmann and Statham. They are not found in the circulating 
blood, as a rule, but they have occasionally been reported in the 
form of intracellular bodies in fatal cases. 

Occxirrence of Bodies. — The bodies may be found in smears 
made from blood obtained by splenic puncture and in the granu- 
lation tissue taken from the ulcers ; also in the mesenteric lymph- 
glands, the bone-marrow, and the liver. Some of these bodies lie 
free, but most of them are intracellular, either in the leukocytes, 
endothelial or splenic cells, and frequently in large masses in 
the macrophages. 

Method of Staining. — The films should be thinly spread, 
after proper fixation, or without, if a form of Eomanowsky stain 
be used, and then stained in the usual way. By Wright's method 
the chromatin appears dark, the cell-body blue, and the re- 
mainder a fainter mauve. 

Blood Changes. — The changes in the blood are those of a 
moderate anemia, associated with a leukopenia and a relative and 



172 ANIIVIAL PAKASITES. 

absolute increase in the number of the large mononuclears. The 
average leukocyte count is about 2000. 

SYPHILIS. 

The SpirocJicBta pallida {Treponema pallidum) derives its 
name from its low refractive power and the difficulty with which 
it takes aniline dyes. It is a very delicate structure^ presenting 
10 to 40 deeip spiral incurvations in the larger specimens or only 
a few in the smaller ones. Its length varies between 4 and 10 
microns and its width does not exceed % micron. The organism 
has been demonstrated in the circulating blood, in the scrapings 
obtained from the chancre, in incised papules, in smears from 
the mucous patches, and in fluid aspirated from the inguinal 
glands. It seems to be easily demonstrable in the blood from a 
splenic puncture, while in the congenital forms it is found in the 
internal organs and in the peripheral blood. A characteristic 
difference between this spirochata and some other t3^pes with 
which it might be confused is that its ends lie above and below 
a longitudinal line drawn through the center of its curvatures, 
while in the other forms the ends lie on the projection of such a 
line. The organism moves in an oscillatory manner about its 
longitudinal axis, its movements being winding, bending, and 
whipping. Schaudinn demonstrated the existence of a flagellum 
at each end, while the other spirochastes have an undulating 
membrane. 

It is in active motion when examined in the fresh, and may 
maintain its activity for days in salt solution, when the tissues 
are kept at 20° to 27° C. (Beer). 

Methods of Obtaining a Specimen for Examination. — The 
surface from which the specimen is to be taken is cleansed with 
alcohol, irrigated with salt solution, and dried. It is then 
scraped with a needle, care being taken to avoid drawing blood, 
but bringing out a good deal of serum. Smears are made from 
the serum, as it is in this that the spirochgete will be found. If 
desired, serum may be drawn from an enlarged gland by means 
of a hypodermic needle and the smears made from this. 



STAINING METHODS. 173 



STAINING METHODS. 



Giemsa Method. — The smear is dried in the air, then placed 
for an hour in absolute alcohol, and then stained for twenty-four 
hours in diluted Giemsa mixture. (For preparation of stain see 
Appendix, page 481.) One drop of Giemsa stain to 1 cubic 
centimeter of the distilled water is the strength used. The 
organism stains a delicate violet-purple color, while the nuclei 
of the leukocytes are of a deep blackish red. The time for stain- 
ing may be materially shortened by adding a few drops of a 
^000 solution of potassium carbonate to the diluting water. By 
this expedient the spirocha^te may be demonstrated in fifteen 
minutes, though for the best results prolonged contact with the 
stain is advised. 

Method of Oppenheim and Sachs. — The smears are dried in 
the air and then, without previous fixation, are flooded with an 
alcoholic solution of carbol-gentian-violet (for preparation of 
stain see Appendix). After flooding the preparation it is 
warmed until it steams for a few minutes only; it is then 
washed gently in running water, dried, and examined. The 
spirochsetes are stained blue. 

Goldhorn Method. — The smears are fixed with pure methyl 
alcohol for fifteen minutes and are then covered with the stain 
(polychrome methylene-blue) for three to five seconds, when the 
excess is drained off. The specimens are then slowly introduced 
into clean water with the film sides down. Keep the slides in 
this position for four or five seconds and then shake in the water 
to remove the excess of the dye. The spirochsetes appear of a 
violet color. This violet tint may be changed to a bluish black 
by covering the specimen with Gram's iodine solution for from 
fifteen to twenty seconds, after which it is washed and dried as 
usual and the examination made with the immersion lens. 

Method of F. C. Wood^ (fifteen minutes) .—The smears 
are fixed in strong methyl alcohol and dried with blotting paper. 
The following stains are then applied in succession : — 

First, a few drops of a /4ooo watery solution of yellow 
water soluble eosin are spread over the smear by means of a 
pipette. Second, four or five drops of a %ooo aqueous solution 

fi Medical News, 1903, Ixxxiil. 248. 



174 ANIMAL PARASITES. 

of methylene azure II. The two colors are thoroughly mixed by 
spreading over the smear and by rocking the cover-glass. This 
mijj:ture is allowed to act for eight minutes. The stain is then 
washed off by a strong stream of distilled water, the preparation 
dried between blotters, mounted, and examined by an oil-immer- 
sion. For mounting use xylol-damimar, as the ordinary Canada 
balsam bleaches these preparations. Usually any precipitate 
which forms can be removed with the stream of water. If espe- 
cially clean and clear specimens are desired, the precipitate may 
be removed by momentary immersion in 95 per cent, alcohol, 
which should be imaajediately washed off with distilled water. 
By this method tlie ispii'oohagtes :appear a light carmine color. 

Method of E. Koll)t.i^~K:olb describes a staining method 
which, he states, takes less than one minute. The technic is 
recommended especially to the general practitioner for its sim- 
plicity and convenience. The stain is a combination of 0.5 
part eosin B. A., 50 parts alcohol, and 40 parts Ehrlich's triacid 
stain (see Appendix for preparation). The stain thus contains 
four dyes ; the mixture should be clear. He prefers oozing serum 
for the test, obtained after a papule is rubbed a little or an 
erosion curetted. The specimen is fixed, a few drops of the 
stain are poured on it, and the whole heated over a flame ; then 
rinsed with water, after which a large amount of a 10 per cent, 
solution of commercial (8 per cent.) acetic acid is poured over it 
carefully two or three times from the edge. The specimen should 
be uniformly pink, while the spirochBetes and bacteria do not 
take the stain, but show up white against the pink background, 
unless the staining is too intense, in which case it is better to 
use a new smear and shorten the staining time. The spirochaetes 
seem to show up better in the edges of the preparation. 

India Ink Method. — Burri advanced this method, which is 
simple and reliable, although some specimens of ink may show 
confusing artefacts, due, according to Barach, to the use of an 
inferior ink. A drop of the fresh serum from the lesion is placed 
at one end of a glass slide and immediately mixed with a small 
drop of Gunther- Wagner India ink (chin-chin liquid pearl ink). 
The mixture is then spread and allowed to dry. The specimen 
is studied with the immersion lens. The whole field is a homo- 

10 Miinch. med. Wochen., June 28, Ivii, No. 26. 



STAINING OF LIVING SPIROCHETES. 175 

geneous brown or black color. The treponema, blood-cells, etc., 
appear as colorless, highly refractile bodies on a homogeneous 
black or brown field. 

Method for Rapid Staining of Living Spirochsetes.— 
Meirowskyii mixes methyl-violet stain with a few drops of 
physiologic salt solution and rubs the colored mixture vigorously 
into the ulcerated primary sore or condyloma. As serum oozes 
it is found to contain the spirochsetes stained a bright violet. 
The depth of the stain depends on the concentration of the 
coloring mixture; it works best when it is of such a strength that 
the liquid envelope of the r^d corpuscles Is.b- deep bluish-violet 
tint. The refringens ass"RS3e a bluish-violet tint, especially 
distiQguished from the bright YiQlat stain of the pallida. 

By the above methods of examination it will usually be 
found that the organisms are most numerous ia moist papules 
and chancres (when the curettage is carried out at the edge of the 
lesion). In scrapings from roseola the search is frequently 
disappointing. The organism should be distinguished from 
S. refringens, 8. dentium, 8. mucosum, etc. 

This section would not be complete without mention of the 
•recent and important researches of N'oguchi, who has succeeded 
in culturing the organism of syphilis. 12 The following is a 
synopsis of his findiQgs : — 

"The standards by which I identify 8piroclicsta pallida in 
cultures are: (1) correct morphology; (2) necessity of the 
presence of sterile fresh tissue in culture medium; (3) strict 
anaerobiosis ; (4) rather faint hazy growth in solid or fluid 
mediums, without any noticeable change in the proteid con- 
stituents; (5) non-production of any offensive odor in culture; 
(6) capability of inciting an allergic reaction on the skin of 
certain cases of syphilis and parasyphilis (so-called luetin re- 
action) ; (7) specific complement fixation with the antipallida 
immune serum of certain serums from human cases of syphilis, 
provided that the antigen is suspended in saline solution and not 
prepared by an alcoholic extraction, and (8) pathogenicity. 
The pathogenicity may be gradually attenuated in course of 



11 Ihid., July 5, Ivii, No. 27. 

12 Jour. Amer. Med. Assoc, Oct. 5, 1912, vol. lix, No. 14. 



176 ANIMAL PARASITES. 

ciiltivation, but the other seven conditions shonld be constantly 
fulfilled. 

"There are some small spirochsete varieties which are dif- 
ficult to differentiate from the pallida. Morphologically, Spiro- 
chceta microdentium and Spirochceta mucosa closely resemble the 
pallida, but both of the former can grow in ascitic agar without 
the addition of fresh tissue and produce an offensive odor. 
Xeither variety binds complement with the antipallida immune 
sermn when used in an aqueous suspension. From the appear- 
ance of the gi'0"wi:h Spirochceta microdentium and SpiroclicEta 
refringens closely resemble the pallida, but they are quite dif- 
ferent in morphologic features. Xeither of these organisms pro- 
duces any odor in culture and the S. refringens grows without 
the fresh tissue." 

Noguchi's Original Technic of Culture. — The syphilitic 
material is first inoculated into the testicle of rabbits where it 
is allowed to develop lesions over a period of from four to six 
weeks. This infected testicular material is then transferred to 
another rabbit and so on until the treponemata are freed of con- 
taminating organisms. The syphilitic testicular tissue is then 
transferred to his special culture medium. 

Preparation of Culture Medium. — Sixteen cubic centimeters 
of serum water, prepared by mixing 3 parts of distilled water 
with 1 part of clear sheep-serum, is placed in a test-tube (20 
X 150 centimeters). A number of similar tubes may be prepared 
These tubes are sterilized by the fractional method at 100° C. 
for fifteen minutes on each of three successive days. Then a 
portion of fresh sterile rabbit testicle is placed in each tube 
which is then incubated at 37° C. for forty-eight hours, after 
which it is examined for sterility. If sterile a layer of sterile 
paraffine oil is run into each tube to obtain anaerobic conditions 
and prevent contamination. These tubes are then inoculated 
from the syphilitic rabbit testicle and cultured at 37° C. 

Detection of Treponema Pallidum in Tissue. — Method 
of Levaditi and Manouelian. This is an improvement over the 
original method of Levaditi and is as follows : — 

Cut pieces of suspected tissue into 1x2 millimeter squares. 
Fix in 10 per cent, formalin for twenty-four hours and harden 
in absolute alcohol for twenty-four hours. Wash repeatedly in 



ANIMAL PARASITES OF MAN. 177 

distilled water. Place the tissue in a Petri-dish and cover with 
silver-p3'ridin solution (see Appendix, page 504). 

Allow to remain in this solution at room temperature for 
four hours and place in incubator at 50° C. for four hours 
additional. Wash in 10 per cent., pyridin solution. Cover for 
four hours at room temperature with a freshly prepared pyro- 
gallol solution (see Appendix, page 500). 

Eemove and dehydrate with absolute alcohol (2 changes), 
clear in xylol and prepare and cut paraffine sections; after 
cutting stain on the slide with 2 per cent. aq. sol. of toluidine 
blue (two minutes) and pass again through alcohol, then oil 
of bergamot and xylol. Mount and examine in the usual way. 
Spirochsete are stained black, groundwork blue. 

Method of No^cM. — ^Cut tissues in 3 x 4 millimeter 
squares, harden in 10 per cent, formalin for several days. 
Transfer without washing to Noguchi's pyridin mixture and im- 
merse for five days at room temperature (see Appendix, 
page 500). 

Wash in distilled water, several changes, for twenty-four 
hours. Transfer to ordinary alcohol (95 per cent.) for three 
days, then wash in distilled water for twenty-four hours. 
Place tissue in an amber bottle and cover with 1.5 per cent. aq. 
sol. silver nitrate for three days at 37° C. (room temperature 
for five days), wash in distilled water for several hours. Pre- 
cipitate the silver by immersion in Noguchi^s pyrogallol solu- 
tion for twenty-four hours (see Appendix, page 500). 

Wash and dehydrate by passing every second day from 80 
per cent, alcohol to 95 per cent, and to absolute alcohol. Clear 
and mount in the usual fashion. Treponema black, neuroglia 
fiber brown (never black), tissues pale yellow to brown. 

Examination by Dark-field Illumination (see Section I, 
page 7). 

ANIMAL PARASITES. 

Temporary parasites include the ectoparasites (epizoa). 
These may inhabit the skin, conjunctival sac, the mouth, the 
nose, and the accessory sinuses. A familiar example of this 
class is the Sarcoptes scabiei, or itch mite. (See page 194, etc.) 

12 



178 ANIMAL PARASITES. 

Most of the permanent or stationary parasites are found in 
the internal organs, and belong to the class of entoparasites or 
entozoa. Many of these parasites, snch as the tenise, ascarides, 
and ankylostoma, inhabit man only when mature; others, of 
which the echinococcus is an example, inhabit man only during 
a certain period of their existence. Thus, man may be either 
the actual or only the intermediate host. Again, for many para- 
sites, such as the Tenia solium and the Tenia saginata, man is 
the only host. Finally, man may also become the host for para- 
sites which, as a rule, select some other animal. Thus, the Cysti- 
cercus cellulosw, common to the pig and the cat, may occasion- 
ally be found in man. 

In this section the classification of Max Braun^s has been 
adopted, and the majority of the descriptions of the parasites 
which follow have been abstracted from that work. 

No attempt has been made to include the rarer forms of 
parasites, as these are considered to be beyond the scope of this 
work. 

CLASSIFICATION OF THE MORE COMMON ANIMAL 
PARASITES OF MAN. 
A. Protozoa. 

Class I. Ehizopodia. — Ameba coli (Loesch). 
Class II. Flagellata (Mastigophora). 

(a) TricJiomonides : 

1. Trichomonas vaginalis. 

2. Trichomonas intestinalis. 

3. Trichomonas pulmonalis. 

( b ) Circomonides : 

1. Lamblia intestinalis. 

2. Trypanosoma. 

Class III. Sporozoa. 
Coccidia: 

1. Coccidium perforans or hominis. 

2. Hemosporidia. 

Class IY. Intusoria. — Balantidium coli or Parame- 
cium coli. 



13 English translation of "The Animal Parasites of Man." (Wood & Co. 
1906.) 



PROTOZOA. 179 

B. Platyhelminthes (Flat worms). 

Class I. Trematoda (Rud). 

1. Fasciola hepaticum syn. distomum hepaticum. 

2. Distomum pulmonale syn. distomum Westermani, 

3. Distomum lanceolatum syn. dicrocelium lanceo- 

latum. 

4. Distomum hematobium syn. bilharzia, syn. schis- 

tosomum hematobium. 

Class II. {Rud.) 

( a ) BothriocepJialoidia : 
Bothriocephalus latus syn. tenia lata. 

(b) Teniidce: 

1. Tenia nana. 

2. Tenia lanceolata. 

3. Tenia solium. 

4. Cysticercus acanthotrias. 

5. Tenia saginata or medioeanellata. 

6. Tenia echinoeoceus. 

C. Nematoda {Thread worms). 

1. Strongyloides (rhabdonema strongyloides) syn. anguil- 

lula intestinalis et stercoralis. 

2. Filaria sanguinis hominis. 

3. Trichocephalus dispar {whip worm). 

4. Trichina spiralis. 

5. Ankylostoma duodenale. 

6. Uncinaria Americana. 

7. Ascaris lumbricoides. 

8. Oxyuris vermicularis. 

A. PROTOZOA. 

The protozoon is a microscopic living organism. It is mono- 
cellular and represents the lowest form of animal life. The 
substance of the body consists of a finely granular, contractile 
protoplasm, which may be mono- or poly- nuclear. The viscid 
hyaline entosarc is capable of motion by expansion and con- 
traction, or by the extension and retraction of pseudopodia, 
cilia or flagella. Propagation takes place by segmentation or 
gemmation. 



180 ANIMAL PARASITES. 

CLASS I. RHIZOPODIA. 

The ameba histolytica produces the well-known amebic 
dysentery. The ameba in man is, however, not confined to the 
intestines, but has been found in the pus of liver abscesses, in 
pleuritic and peritoneal exudates, in the mucous membranes, 
and in tumors of the urinary bladder. 

Characteristics. — This organism is an ameboid body meas- 
uring from 20 to 30 microns in diameter. It is composed struc- 
turally of a clear protoplasmic outer portion, ectosarc, and a 
finely or coarsely granular central portion, entosarc, which usu- 
ally shows a number of clear vacuoles and one or more nuclei 
(Plate yi, a). When living it shows active ameboid movements 
which are greatly increased if the organism is kept warm. In 
the living state the cell frequently includes foreign bodies, such 
as bacteria, pigment granules, and fragments of blood-corpuscles 
and other cells. 

Locomotion is accomplished by irregular extension and re- 
traction of pseudopods, which are thrown out from the periphery. 
These pseudopods are at first composed of the clear outer por- 
tion, which, as the projection gradually increases, includes the 
central granular zone. When surrounded by unfavorable envi- 
ronment, the organism undergoes a form of change known as 
the encysted state. In this state the body becomes spherical 
and the outer wall thickened, while the division of the cell into 
two portions is lost^ the whole becoming uniformly granular. 

Method of Examination. — The fecal discharges should be 
caught in a warm receptacle and immediately transferred to the 
laboratory for examination. If it is necessary to keep the 
specimen for a short time, this may be accomplished by imme- 
diately placing the specimen in a thermostat at body tempera- 
ture, where it should remain until transferred to the warm stage 
of the microscope. Preservation of specimens for more than a 
few hours is unsatisfactory, because even under the favorable 
circumstances of heat and moisture obtaining in the thermostat, 
motility is rapidly lost, and at the expiration of twenty-four 
or thirty-six hours the organisms are no longer discoverable. 

The Warm Stage. — A convenient method of maintaining 
a warm stage for examination and study of this organism is as 



PLATE VI. 




Intestinal Parasites of Man. {Redran^vn from Max Braun) 



a, Amebae coll. b, Trichomonas vaginalis. 
d, Lamblia Intestinalis. ^, Tripanosomes 



r, Trichomonas intestinalis. 
^ Coccidium hominis. 



FLAGELLATA. 181 

follows: A flat strip of copper or of brass, three inches wide 
by six or eight inches long, is perforated by a half- or three- 
quarter inch aperture, situated in the center of one end at a 
distance of about one inch from the free margin. The stage 
of the microscope should be covered with several thicknesses of 
asbestos paper or felt, upon which the metal sheet is clamped 
so that the openings in the stage and in the copper-strip coin- 
cide. A Bunsen burner or alcohol lamp is adjusted under the 
outer extremity of the strip, so that when the metal has attained 
its maximum heat the portion immediately surrounding the 
aperture is maintained at about body-temperature. A specimen 
placed upon a slide, in position for examination, will maintain 
the motility of the ameba for a number of hours. 

It will be found convenient to examine the spread, first by 
the low power, then, after locating a suitable portion of the 
slide, the higher objective may be swung in and the organisms 
studied in detail. The important aids to successful search are 
a thin and uniform spread of liquid feces, and a low, slightly 
oblique illumination. The microscope should be as near the 
patient as possible. A high rectal tube is passed, and the fecal 
matter, often containing blood or mucus, found in the eyelets of 
the tube is transferred to a warm slide, covered with a warm 
cover-slip, and immediately studied. In case it is necessary to 
carry the tube to a laboratory, this is best done by arranging two 
pans like a chafing-dish, the lower one containing water at about 
100° ¥., the upper one resting on the water, and containing the 
tube. Before the diagnosis of amebse is justified, one must find 
unicellular organisms, showing actively motile pseudopodia, acd 
moving from place to place by the activity of the pseudopodia. 

CLASS II. FLAGELLATA. 
a. Trichomonides. 

1. Trichomonas Vaginalis (see Plate YI, /;). — The form 
of the body is very variable, elongated, fusiform or bean-shaped. 
It is ameboid. The length varies between 0.015 and 0.025 milli- 
meter in length, by 0.007 to 0.012 in breadth. The posterior ex- 
tremity is drawn out into a point and is about one-half the 
length of the remainder of the body. The cuticle is very thin 
and the body-substance finely granular. At the anterior ex- 



182 ANIMAL PARASITES. 

tremity tbere are three or four flagella, which are of equal length, 
and which,' are firmly united at their base, but which easily fall 
off. There is an undulating membrane which moves spirally 
across the body, arising from the base of insertion of the flagella 
and terminating at the base of the caudal process. The nucleois 
is vesicular, elongated, and situated in the anterior extremity. 
Propagation occurs by division. 

2. Trichomonas Intestinalts (see Plate A^I, c). — Some 
authors believe this organism to be identical with the TricJiomo- 
nas vaginalis. It is described as being 0.01 to 0.015 millimeter 
in length; the posterior extremity terminates in a point with a 
row of cilia. This organism has been found in the urethra, the 
vagina, the large and small intestines, the stomach, and in the 
oral cavity. 

3. Trichomonas Pulmonale. In all probability this is 
identical with the preceding. 

b. Cercomonides. 

1. Cercomonas Intestinalis or Lamblia Intestinalis 
(see Plate VI, d). — ^Leugth, 0.01 to 0.02 millimeter, and width, 
0.005 to 0.012. The flagella are of about equal length (0.009 to 
0.014 millimeter). The body is-finely granular, and has a very 
thin cuticle, which does not entirely prevent changes in the form 
of the body. The very motile, tail-like appendages in the frontal 
plane are flattened; thei evacuation at the anterior extremity 
which is directed obliquely forward and with its border project- 
ing backward. The anterior pair of flagella arise from the ante- 
rior edge of the peristome ; the lateral and median from the pos- 
terior edge, whereas the tail flagella are inserted at the pos- 
terior end. The anterior flagella appear to be connected with the 
nucleus. The nucleus is dumb-bell shaped and has a nucleolus in 
each half, and lies anteriorly in the part of the body which is ex- 
cavated. This organism has encysted stages. These cysts are 
oval and measure .01 by .007 millimeter. They are surrounded 
by a fairly thick hyaline layer through which the outline of the 
creatures can sometimes be seen quite distinctly. 

Trypanosoma. — The Trypanosome^^ has a more or less 
spindle-shaped body, along one border of which runs an undulat- 



14 E. R. Stitt : "Practical Bacteriology," etc., 1909. 



SPOROZOA. 183 

ing membrane. There is a nucleus and a blepharoblast, the 
latter being located anteriorly as a chromatin staining dot or 
rod. From this blepharoblast the flagellum proceeds posteriorly 
bordering the undulating membrane and projecting freely 
be3^ond the posterior end. The nucleus is larger, nearer the 
posterior end, and does not stain so intensely as the bleph- 
aroblast. 

T. Gamhiense. — ^This is the trypanosome causing human 
trypanosomiasis, the later stage of which is known as sleeping 
sickness. It is from 17 to 28 ^^ long, and from 1.5 to 2 ^ wide. 

It is very difficult to distinguish the human trypanosome 
from some of the other pathogenic ones by staining methods. 

It is present in the blood, usually in exceedingly small numbers, 
and in the lymphatic glands of patients. It is by puncture of the 
glands that we have the best means of finding the parasites. The 
parasite stains readily with Wright's stain. The transmitting agent 
is the Glossina palpalis. It is not known whether this occurs by direct 
or indirect transmission. At any rate, no tsetse, no trypanosomiasis. 

CLASS III. SPOROZOA. 
Coccidia. 

1. CocciDiuM HoMiNis OR Pehforans. — This organism is 
oval. The fertilized sporont stage is the oocyte (see Plate YI, /), 
and measures .024 to .035 by .002 to .014 millimeter. It is sur- 
rounded by an integument with a double outline which has an 
opening at the pointed pole. The plasm, which is somewhat 
coarsely granular, entirely fills the integument or is gathered 
together in a rounded central mass. The coccidia are evacuated 
from the bowel in this stage, and sporulate in the open within 
two or three weeks. The fully developed spores are of a broad 
fusiform shape and measure .012 to .015 millimeter in length by 
.007 millimeter broad. They each contain two sporozoites, broad 
at one* end and pointed at the other, forming a bent dumb-bell 
shaped body. The chromatin mass is called the centrosome, and 
the extremity of the body which encloses this, is the anterior 
extremity. 

A granular residual body lies in the concavit}^ This or- 
ganism is found in the intestinal tract, where it may give rise 
to violent auto-infection and chronic diarrhea. 

2. HsMOSPORiDiiE. — These organisms are the cause of ma- 
laria and have been described in anotlier section (see page 159). 



184 ANIMAL PARASITES. 

CLASS IV. INFUSORIA. 

Balantidium Coli or Paramecium Coli. — The dody is 
oval, .06 to 1.0 millimeter in length, by .05 to .07 millimeter 
in width. The peristome is frmnel-shaped or contracted, the 
anterior end being either blunt or pointed. The ecto- and ento- 
sarc are distinctly separate, the latter granular, and containing 
drops of fat, mucus, starch granules, bacteria, and occasionally 
leukocytes and erythrocytes. There are usually two contractile 
A^acuoles. The anus opens at the posterior extremity, and the 
organism contains a macro- and a micro-nucleus. It occurs in 
the large intestines of man. 

B. PLATYHELMINTHES (Flat Worms). 

CLASS I. TREMATODES. 

1. DiSTOMUM Hepaticum (Liver fiiike). — Length, 20 to 
30 millimeters; breadth 8 to 13 millimeters. The head-cone 
is 4 to 5 millimeters long, and sharply demarcated from the 
posterior part of the body. Spines in alternating transverse 
rows, and extending on the ventral surface to the posterior bor- 
der of the tested, and on the dorsal surface not quite so far. The 
spines are smaller on the head-cone than on the posterior part 
of the body, where they are discernible by the naked eye. The 
suckers are hemispherical and near each other. The oral sucker 
is about 1 millimeter, and thd ventral about 1.6 millimeter in 
diameter. The pharynx, which includes almost the entire esoph- 
agus, measures .7 millimeter in length and .4 millimeter in 
breadth. The intestine bifurcates in the head-cone, and the 
branches are furnished with blunt sacs directed outwardly. The 
ovary is ramified and is situated in front of the transverse 
vitello duct. The shell-glands lie near the ovary and are in the 
median line. 

The ova are yellowish-brown, oval, with cap-like lid. They 
measure .130 to .145 by .07 to .09 millimeter. The average 
size is .132 by .08. The liver fluke is an inhabitant of the bile- 
ducts of man. 

2. DiSTOMUM Pulmonale or Distomum Westermanni. — 
The body is of a faint, reddish-brown color and plump oval 
ghape^ with the ventral surface a little flattened, This organism 



PLATYHELMINTHES. 185 

measures 8 to 10 millimeters in length by 4 to 6 millimeters in 
thickness. It possesses two sucJcers of equal size (.75 milli- 
meter) . 

The eggs are oval brownish-yellow, with a fairly thin shell 
and measure .0875 to .1025 millimeter in length, by .052 to 
.075 millimeter in breadth, the average being .0935 by .057 milli- 
meter. Their location is usually in the lung, but they may 
enter the blood-vessels and be carried to another part of the 
body. 

3. DiSTOMUM Lanceolatum or DicrO'Celium Lanceo- 
LATUM. — In the fresh condition this is a yellowish-reld organism, 
the bod^i/ is flat, almost translucent, with a conical neck at the 
level of the ventral sucker. This point is marked by a shallow 
constriction. The length and breadth vary according to the 
amount of contraction, being usually 8 to 11 millimeters by 
1.5 to 2.0 millimeters. The suclcers are about one-fifth of the 
body-length distant from each other, and of about dqual size 
(.23 by .25 millimeter). 

The eggs are oval with a sharply defined operculum at the 
pointed pole. They measure .030 by .011 millimeter, and occur 
in the feces. The parasites reside in the liver. 

4. DiSTOMUM Hematobium or Bilharzia. — The male is 
whitish and measures 12 to 14 millimeters in length, but is 
already mature when 4 millimeters long. The anterior end is 
6 millimeters or a little more in length ; the suckers are situated 
near each other; the oral sucker is infundibulif orm ; the dorsal 
lip being a little longer than the ventral. The ventral sucker 
is slightly the larger, and is pedunculated. A little behind the 
ventral sucker the body broadens to a width of 1 millimeter, 
decreasing, however, in thickness. The lateral edges curl ven- 
trally, so that the posterior part of the body is almost cylin- 
drical. The posterior end is somewhat attenuated, and the 
dorsal part of the posterior extremity of the body is covered 
with spinous papilli. 

The females are filiform, about 30 millimeters in lengtli. 
pointed at each end and measuring .25 millimeter in diameter. 
The color of the body varies according to the contents of the 
intestine (posteriorly they are dark-brown or black). The 
cuticle is smooth except in the suckers, where there are very 



186 ANI]VIAL PARASITES. 

delicate spines, and at the tail end where there are larger spines. 
The anterior part of the body measures from .2 to .3 millimeter 
in length. 

The eggs are fusiform and much dilated in the middle. 
They have no lid and are provided with a terminal spine at the 
posterior extremity. The eggs measure 0.12 to 1.12 millimeters 
in length, by .05 to .073 millimeter in breadth. They are yel- 
lowish in color, slightly transparent, and provided with a thin 
shell. The spine may sometimes be absent. The eggs apparently 
vary greatly in size. The organism lives in the portal vein and 
its branches. 

CLASS II. 

a. Bothriocephaloidia. 

1. BoTHRTOCEPHALis Latus OR Texia Lata. — Length 
from 2 to 9 or more millimeters. Color, 3^ellowish gxay: after 
lying in water lateral areas become brownish and the rosette 
of the uterus brown. The liead is almond-shaped, 2 to 3 milli- 
meters in length. Its dor so- ventral axis is longer than its trans- 
verse diameter; the head is therefore generally fiat, concealing 
the suctorial grooves at the borders. The neck varies in length 
according to the degree of contraction, and is very thin. There 
are from 3000 to 4500 proglottides. Their breadth is usually 
greater than their length, but in the posterior third of the 
body they are almost square, while among the very oldest some 
may be longer than they are broad. 

The eggs are large with brownish shells and small lids. 
They measure .068 to .071 by .045 millimeter. The proglottides 
near the posterior extremity, of the worm are frequently eggless. 

b. Tenildae. 

1. Texia Xana. — The worm is 10 to 15 millimeters in 
length, and .5 to .7 millimeter in breadth. The liead is globular 
and is from .25 to .30 millimeter in diameters. The rostellum 
has a simple crown consisting of 24 to 30 hooks, which are only 
.014 to .018 millimeter in length. The neck is moderately long. 
The proglottides are very narrow, about 150 in number, .4 to .9 
millimeter in breadth, by .014 to .030 millimeter in length. 



PLATYHELMINTHES. 187 

The eggs are globular or oval, and measure .030 to .048 
millimeter. The oncospheres measure .016 to .019 millimeter 
in diameter. 

The worm lives in the intestines; the ova, and proglottides 
are found in the feces. 

2. Tenia Lanceolata or Hymenolepis Lanceolata. — 
The parasite measures, 30 to 130 millimetelrs in length, and 5 to 
18 millimeters in breadth. The head is globular and very small, 
the rostellum is cylindrical with a crown composed of eight 
hooks (0.031 to 0.035 millimeter in length). The neck is very 
short. The segments increase gradually in breadth, but vary 
little in length. 

The ova have three envelopes and are oval, measuring 0.050 
by 0.035 millimeter. The external envelope is membranous and 
much wrinkled, the middle one is thick, and the internal one 
very thin. 

3. Tenia Solium or Tenia Vulgaris. — The average 
length of the entire tapeworm is about 2 to 3 meters, but 
may be more. The head is globular, 0.6 to 1.0 millimeter in 
diameter. The rostellum is provided with a double row of hooks, 
twenty-two to thirty- two in number; large and small hooks 
alternate regularly. Thei length of the largest hooks is 0.16 to 
0.18 millimeter, of the small ones 0.11 to 0.14 millimeter. The 
average number of proglottides is 800 to 900; they increase 
very gTadually in size. At about 1 millimeter behind the head 
they are square and have the generative organs fully developed. 
Segments sufficiently mature for detachment measure 10 to 12 
millimeters in length, by 5 to 6 millimeters in breadth. The 
fully developed uterus' ^consists of a median trunk with seven to 
ten lateral branches on each side, some of which are again 
ramified. 

The eggs are oval, the egg-shell very thin and delicate. The 
embryonal shell is very thick with radial stripes ; it is of a pale- 
yellow color, globular, aud measures 0.031 to 0.036 millimeter 
in diameter. The oncospheres with six hooks are likewi^:e globu- 
lar, and measure 0.02 millimeter in diameter. 

4. Cystice:rcus Acantiiotrias. — This' resembles the cii^ti- 
cercus cellulose in form and size, but carries on the rostellum a 
triple crown each cousisting of fourteen to sixteen hooks which 



188 ANIMAL PARASITES. 

differ from the hooks of the cysticercus^ cellulose or of the tenia 
solium by the greater length of the posterior root process and 
the more slender form of the hooks. The large hooks measure 
0.153 to 0.196 millimeter, the medium-size hooks 0.114 to 0.14, 
and the small ones 0.063 to 0.07. 

5. Tenia Saginata or Tenia Mediocanellata. — The 
length of the entire worm averages 4 to 8 to 10 meters and 
more, even up to 36 meters. The head is cuboid in shape, 1.5 
to 2 millimeters in diameter. The suckers are hemispherical 
(0.8 millimeter), and are frequently pigmented. There is a 
sucker-like organ in place of the rostellum, and this is also 
frequently pigmented. The neck is moderately long and about 
half the breadth of the head. The proglottides average more 
than 1000, and gradually increase in size from the head back- 
ward. The detached mature segments are exactly like pumpkin- 
seeds — they are about 16 to 20 millimeters long, by 4 to 7 milli- 
meters broad. There are twenty to thirty-five lateral branches at 
each side of the uterus, and these often again ramify. 

The eggs are more or less globular, the egg-shell frequently 
remains intact, and carries one or two filaments. The embryonal 
shell is thick, radially striated, transparent and oval. It is 0.3 to 
0.4 millimeter in length, by 0.02 to 0.03 millimeter in breadth. 

This worm in the adult condition dwells exclusively in the 
intestinal canal of man. The corresponding cysticercus occurs 
ill the ox and steer. 

6. Tenia Echinococcus. — This worm measures 2.5 to 5 or 
6 millimeters in length; the head is 0.3 millimeter in breadth, 
and has a double row of twenty-eight to fifty booklets on the 
rostellum. The size and form of these booklets vary. The 
larger ones are 0.040 to 0.045 millimeter in length, the smaller 
ones are 0.030 to 0.038 millimeter. The suckers measure 0.13 
millimeter in diameter. The neck is short, behind which there 
are only three or four segments, the posterior of which is about 
2 millimeters in length and 0.6 millimeter in breadth. The 
ovary is horse-shoe shaped, with the concavity directed back- 
ward. The median trunk of the uterus is dilated when filled with 
eggs, and instead of lateral branches has lateral protuberances. 
It is not uncommon for the eggs to form local heaps. The em- 



NEMATODES. 189 

hrijonal shell is moderately thin with radiating) fibers, is almost 
globular, and measures 0.030 to 0.036 millimeter in diameter. 

The mature parasite lives in the small intestine of the 
domestic dog and the wolf, and from them, the dog chiefly, they 
are transmitted to man. 

Diagnosis of Cestodes. — The microscopic examination of 
the feces should never be neglected when the presence of tape- 
worm is suspected. Often by careful, frequently repeated exami- 
nations, insistent symptoms, referable to the digestive tract, the 
nervous system or the general nutrition, may be cleared up by 
the finding of segments or ova in the feces. 

As the uterus of the tenia has no exit, the eggs can only 
find egress when the mature proglottide is injured. In the case 
of the tenia saginata the discharge of eggs is almost the rule. 
The proglottides when discharged are usually without eggs. The 
eggs of the solium and the saginata are only distinguished by 
their size. 

In examining the stools for evidences of tapeworms, one 
must be careful not to confound remnants of undigested food, 
mucous casts, and shreds of tendon with the proglottides. The 
proglottides, -after being soaked in water, assume their character- 
istic form. As a rule the microscopic determination of ova is 
a more certain means of diagnosis than the macroscopic seg- 
ments. 

To determine from the shape of the proglottide which 
variety of worm is present, it is advisable to fix the segment 
between two glass slides. The proglottide of the tenia solium is 
more delicate and more transparent than the tougher segments 
of the tenia saginata. In the former the branching uterus is 
more plump, and the number of lateral twigs are from seven to 
ten, while the uterus of the tenia saginata shows from twenty to 
thirty or more. 

C. NEMATODES (Thread Worms). 

Strongyloides Intestinalis or Anguillula Intesti- 
NALis ET Stercoralis. — 1. (a) The parasitical generation 
(anguillula intestinalis) measures 2.3 millimeters in length by 
0.034 millimeter in breadth. The cuticle is finelv transverselv 



190 ANIMAL PAEASITES. 

striated. The mouth is surrounded by four lips, the esophagus 
is almost cylindrical and is a quarter the length of the body. 
The anus opens just in front of the pointed posterior extremity. 

The eggs measure 0.050 to 0.58 millimeter in length, and 
0.030 to 0.034 in breadth. 

(b) The free-living generation (anguillula stercoralis) is sex- 
ually differentiated. The body of the male is cylindrical, 
smooth, somewhat more slender at the anterior extremity, and 
pointed at the tail end. The mouth has four lips and the 
esophagus a double dilatation. The males measure 0.7 by 0.035 
millimeter, and carry the posterior extremity curled up. The 
two spicules are small and much curved. The females measure 
1.0 millimeter in length or a little more, 0.05 millimeter in 
breadth. The tail end is straight and pointed. The yellowish 
thin-shelled ova measure 0.07 millimeter in length by 0.045 
millimeter in breadth. 

2. FiLARiA Saj^guinis Hominis (filaria hancrofti or filaria 
nocturna). — The male is colorless and measures 40 millimeters 
in length and 0.1 millimeter in diameter. The cephalic ex- 
tremity is a little thickened, the posterior extremity is bent and 
rounded, but is not twisted cork-screw like. The anal orifice 
opens 0.138 millimeter in front of the posterior border. The 
female is brownish, 7.8 to 8.0 millimeters in length and 0.21 to 
0.28 millimeter in breadth. The cephalic and caudal extremi- 
ties are rounded. Almost the entire body is occupied by the 
two uteri, from which the larvae emerge early. The length of 
the larvce average 0.13 to 0.3 millimeter, their breadth 0.007 to 
0.07 millimeter. They are surrounded by a delicate protective 
investing membrane which is not quite close to them. 

The l3^mphatic vessels in various parts of the body are 
doubtless the normal habitat of the adult worms, but these 
have also been found in the left ventrice of the heart. The 
young ova, by means of the lymph-stream, reach the blood and 
are distributed with it through the body. They also pass through 
the vessel walls and may be found in the fluid of the glands of 
the body. The larvae are first found in infected patients only in 
specimens of blood that have been taken after sunset. Their 
number increases considerably until after midnight, and after 



NEMATODES. 191 

that time begin to diminish. From mid-day until evening no 
filariag are found in the peripheral blood. 

3. Trichocephalus Dispar or Ascaris Trichiura. — The 
male measures 40 to 45 millimeters in length, the spiculnm is 
2.5 millimeters long and lies within a retractile pouch beset with 
spines. The female measures 45 to 50 millimeters in length, of 
which two-fifths appertain to the posterior part of the body. 
The ova are barrel-shaped and have a thick, brown shell which 
is perforated at the poles. Each opening is closed with a light- 
colored plug. The eggs measure 0.05 to 0.054 millimeter in 
length and 0.023 millimeter in breadth. They are deposited 
before segmentation. This worm usually lives in the cecum of 
human beings, and is occasionally found in the vermiform ap- 
pendix, in the colon, and exceptionally in the small intestine. 
Usually only a few are present, and they do not cause any par- 
ticular disturbance. 

The development of the eggs is completed in water or in 
moist soil, and occupies a longer or shorter period according to 
the season. The eggs and larvae possess great powers of resist- 
ance, and have been known to remain as long as five years in 
the egg-shell without losing their vitality. 

4. Trichiis^a Spiralis. — The male measures 1.4 to 1.6 mil- 
limeters in length and 0.04 millimeter in diameter. The ante- 
rior part of the body is narrowed, the orifice of the cloaca is 
terminal and lies between the two caudal appendages; behind 
there are four papillae. The female measures 3 to 4 millimeters 
in length and 0.06 millimeter in diameter; the anus is terminal. 
Trichina spiralis occupies in its adult stage the small intestines 
of men and of various mammals, including the domestic rat, 
domestic pig and domestic dog. 

History of Development of Trichina Spiralis. — 
Shortly after entering the intestines the encysted trichinae 
escape from their capsules, and then enter the duodenum and 
jejenum where they become adult. During this period they 
do not greatly increase in size. The males grow from 0.8 to 1.0 
millimeter, the females from 1.5 to 1.8 millimeters. Soon after 
copulation, which takes place in the course of two days, the 
males die off, and the females, which soon attain the length of 
3.0 to 3.5 millimeters, either bore more or less deeply into 



192 ANIMAL PAKASITES. 

the villi or penetrate the mucous membrane and enter the lym- 
phatic spaces. Here they deposit their young which, according 
to Leuckart, average at least 1500. The migrations are mostly 
passive, the larvge being carried along by the h'mph-stream or 
by the circulating blood. The young brood is distributed 
throughout the entire body, but the conditions necessary to its 
further development, are found only in the transversely striated 
muscle. On the ninth or tenth day after infection the first 
trichinae have reached their destination, but further invasions 
are constantly taking place. Two or three weeks after infec- 
tion the spirally rolled up trichina have grown to 0.8 to 1.0 
millimeter, and in their vicinity the muscle fibers are swollen. 
The capsule is formed by the inflamed connective tissue pro- 
ducing the cystic membrane. The cysts are lemon-shaped and 
usually lie with their longitudinal axis in the direction of the 
muscle fibers. On an average they measure O.-i millimeter in 
length by 0.25 millimeter in breadth. 

5. AXKYLOSTOMA DUODEXALE OR UXCIXARIA DUODENALE. 

— The body is cylindrical, attenuated anteriorly, and of a 
slightly reddish color. In the oral cavity on the ventral surface, 
close behind the orifice, are four hook-like teeth directed back- 
ward; on the dorsal surface there are two teeth directed for- 
ward. The males measure 8 to 10 millimeters in length, and 0.4 
to 0.5 millimeter in breadth. The bursa has two large lateral 
and one small dorsal alar processes. The females measure 12 to 
18 millimeters in length, and the caudal extremity has a small 
spine. The eggs are elliptical and have thin shells; they meas- 
ure 0.032 to 0.0-i5 millimeter in breadth, and 0.055 to 0.065 
millimeter in length. 

The ankylostoma duodenale lives in the duodenum, and 
may rarely be found in the first part of the jejunum. 

6. Uncixaria Americana can readily be distinguished 
from the preceding worm. It is shorter and more slender. The 
male worm measures from 7 to 9 millimeters in length by 0.3 
to 0.35 in diameter; the female 9 to 11 millimeters in length by 
0.4 to 0.45 millimeter in diameter. The buccal capsule is much 
smaller, and presents an irregular border; instead of four ven- 
tral hook-like teeth, it is provided with a vertical pair of promi- 
nent semilunar plates similar to those of a dog hook-worm. 



NEMATODES. I93 

The pair of dorsal teeth is likewise represented by a pair of 
slightly developed ehitinous plates of the same nature. 

The eggs are larger than in U. Duodenale ; they measure 64 
to 75 micromillimeters by 36 by 40 micromillimeters in breadth. 
So far this worm has been found only in man; its anatomical 
habitat is the small intestine. 

7. AscARis LuMBRicoiDES. — The coloring in the fresh 
stage is reddish- or grayish-yellow. The body is of an elongated 
spindle shape and the dorsal oral papillae carries two papillae 
of sense and the two ventral oral papill£e are papillae of sense. 
The male measures from 15 to 25 centimeters in length and 
about 3 millimeters in breadth. The posterior extremity is 
conical and bent ventrically into a hook. The spicules measure 

2 millimeters in length and are curved and broadened at their 
free ends. On each side of the orifice of the cloaca are seventy 
to seventy-five papillae, of which seven pairs are post-anal. 

The female measures 20 to 40 centimeters in length 
and about 5 millimeters in diameter, the posterior extremity is 
conical and straight. The vulva is at the border between the 
middle and posterior thirds of the body, from which the two 
uterine tubes pass straight to the posterior end of the body. 
The convoluted ovaries measure ten times the length of the body. 

The ova are elliptical with a thick transparent shell and an 
external coating of albumin which forms protuberances. The 
ova measure 0.05 to 0.07 millimeters in length and 0.04 to 0.05 
millimeter in breadth; they are deposited before segmentation. 
The albuminous coating is stained yellow by the coloring matter 
of the feces. 

This worm is one of the most frequent parasites of man, 
and is distributed over all parts of the world. 

8. OxYURis Vermicularis or Ascaris Vermicularis. — 
Color, white; the attenuated cuticle forms swellings at the 
anterior end which extend some distance back along the middle 
of the ventral and dorsal surfaces. There are three small re- 
tractile labial papillae around the mouth. The male measures 

3 to 5 millimeters in length, and shortens on death. The poste- 
rior extremity of the body is rolled ventrally and presents pa- 
pillae. The female is 10 millimeters in length and 0.6 milli- 
meter in diameter. The anus is about 2 millimeters in front 

13 



194 ' ANIMAL PARASITES. 

of the tip of the tail; the vulva is in the posterior third of the 
body. The eggs are oval, thin-shelled, and measure 0.05 by 
0.02 millimeter. They are deposited with embryos already de- 
veloped, and are seldom found in the feces. 

TEMPORARY PARASITES. 

PARASITES OF THE SKIN. 
Arthropoda. 

These are bilaterally symmetrical, segmented animals whoso 
segments do not correspond. The segments are often more or 
less fused, thus forming special body-regions which may them- 
selves be more or less fused together as well. The arthropods 
commonly reproduce by ovulation, the development of the 
embryo to the adult often showing more or less complicated 
metamorphoses. The true parasitic forms of the arthropoda 
thus far met in man are limited to the arachnoids and insects. 

Arachnoidea. 

Sarcoptes or Acarus Scabiei (the Itch Parasite). — This 
parasite is oval in shape, is provided with horns and bristles (see 
Fig. 42), and is barely visible to the naked eye. The male 
measures from 0.2 to 0.3 millimeter in length by 0.145 to 0.19 
millimeter in breadth; the female is somewhat larger, showing 
a length of 0.33 to 0.45 m.illimeter and a breadth of 0.25 to 
0.35 millimeter. 

The female lies at the end of a burrow in the epidermis, in 
situations where the skin is most delicate, as between the fingers, 
at the elbows, under the knees, and in the groin. In this bur- 
row, which varies from a few millimeters to a centimeter m 
length, the female deposits her eggs, after which she dies. The 
eggs hatch in from four to eight days, and in about fourteen 
days the larvae are sufficiently matured to make their own bur- 
rows. The disease is communicated either by the clothing or by 
personal contact. To demonstrate the parasite, the burrow is 
opened with a needle and the female is pressed out on a slide, 
which is then covered and examined. 

Demodex FoUiculoruin. — This parasite is very small, vary- 
ing in length from 0.3 to 0.4 millimeter. It is somewhat cylin- 



INSECTA. HEMIPTERA. 195 

drical, tapering to an obtuse point at the posterior end. This 
parasite has its habitat in the sebaceous follicles, especially of the 
face and nose. 

Leptus Autumnalis (Harvest Bug). — This is a minute red 
parasite, from 0.3 to 0.5 millimeter long, which has three pairs 
of legs, with a row of bristles upon its back and belly. It pre- 
vails in summer on grass and plants and attaches itself to the 
skin of man by its booklets. 




Fig. 42.— Sarcoptes Scabiei. 



Insecta. Hemiptera. 

Pediculus Capitis (Head Louse). — The male is from 1 to 1.5 
millimeters long; the female is 1.8 to 2 millimeters long. The 
color of the parasite varies somewhat with the race of its host. 
In the Caucasian it is gray with a dark border ; in the Negro and 
Chinaman it is much darker in color. The eggs are 0.6 milli- 
meter in length and are attached to the hairs, forming the so- 
called "nits.^^ These nits are whitish, oval masses which are 
easily visible. This parasite, while usually found upon the hair 
of the head, may be found in other portions of the body. 

Pediculus Vestimenti (Body Louse). — This parasite is con- 
siderably larger than the former, being from 2 to 5 millimeters 
long and whitish gray in color, the back part of the body being 
wider than the thorax. The antennae are longer than those of 
the head louse. The eggs are from 0.7 to 0.9 millimeter in 
length, about 70 being laid by each female. This parasite is 
found upon the clothing, in which it deposits its eggs, especially 
about the neck, back, and abdomen. 



196 ANIMAL PARASITES. 

Pediculus Pubis (Phthirius Inguinalis, or Crab Louse). — 

This parasite is smaller than the head louse (see Fig. 43), 
grayish yellow or gray white in color, the male being from 0.8 
to 1 millimeter in length, the female about 1.12 millimeters in 
length. The eggs are pear-shaped, from 0.8 to 0.9 millimeter 
in length and from 0.4 to 0.5 millimeter in breadth. 

This parasite infests the parts of the body covered by the 
shorter hairs, such as the pubis, axilla, eyebrows, and chest. 

Cimex Lectularius (Acanthia Lectularia, or Bedbug). — 
While, strictly speaking, the bedbug is not a parasite of man, yet 
as its habitat is the bed, bedding, and walls of the sleeping 
apartments of man, it may be considered as indirectly parasitic. 




Pig. 43.— Pediculus Pubis. 



It usually emerges at night from its lodging for the purpose of 
securing its nourishment in the blood of its victims. 

This parasite is reddish brown in color, oval in shape, from 
4 to 5 millimeters in length and 3 millimeters in breadth. These 
insects, if crushed between slides or as more usual between the 
hand and a part of the victim's body, have a characteristic odor 
very much resembling kerosene. The blood is drawn from the 
victim by means of a long proboscis. The eggs are approxi- 
mately 1.12 millimeters in length and require about eleven 
months for their development to the sexually ripe insect. These 
eggs are retained in the crevices of the bed, floors, furniture, wall- 
paper, and other parts of the dwelling, so that the complete 
removal of these eggs and parasites is a matter of some difficulty. 

That these insects have more or less importance from the 
standpoint of transmission of disease from one person to another 
must be remembered. Individuals vary in their susceptibility to 
the bite of the bedbug, some being indifferent to it, while others 
are markedly affected by it. 



VEGETABLE PARASITES. 197 

Diptera. 

Pulex Irritans (Common Flea). — The male is from 2 to 2.5 
millimeters in lengthy the female as much as 4 millimeters. It 
is a red or brownish-red insect, having a laterally compressed 
body, an oral rostellum, serrated soft mandibles, a tongue 
sheathed in an inferior labium, and a pair of labial four-jointed 
palpi. Each of the triple segments of the thorax bears a pair 
of five-Jointed double-clawed legs. The female deposits her 
eggs, not on the human being, fortunately, but in the fissures, 
crevices, or holes of garments or furniture which may be 
accessible. 

Pulex Penetrans (Sand Flea, or Jigger) . — This parasite is a 
minute, brownish-red, egg-shaped insect which penetrates the 
skin of man. The female is the infecting insect and produces 
painful irritation and even suppuration. 

VEGETABLE PARASITES. 

Achorion Schonleinii. — This organism is the cause of the 
disease known as favus or tinea favosa. This fungus invades 
the root sheaths, the bulbs, and the shafts of the hair filaments of 
the scalp, but it also occurs upon the ^^non-hairy'^ portions of 
the skin and upon the nails. The spores gain access to the 
deeper layers of the skin and develop around the hair-shaft, 
forming a characteristic yellowish, cup-shaped crust which has a 
peculiar, mouse-like odor. 

In searching for this parasite, a favus crust is softened by 
the addition of a few drops of water or dilute sodium hydrate 
solution and placed upon a slide and examined with the high- 
power dry lens. The hairs may also be examined in the same 
manner or may be stained by methods outlined in the discussion 
on Tinea tricJiophytina. 

The mycelial threads appear as narrow, flattened, ramifying, 
short or elongated, linear cells or tubes, which may be simple 
and empty, or be divided more or less regularly by transverse 
partition walls transforming the longer and simpler into shorter 
and compound cells (see Fig. 44). The latter often contain in 
their cavities sporules clinging to either side, in which case the 



198 



VEGETABLE PARASITES. 



mycelial threads are termed sporophores. The conidia are en- 
capsulated or are strung together like the beads of a necklace, 
and appear as round, oval, angular, or very irregularly con- 
toured bodies. These mycelial threads branch at right angles; 
the spores measure from 3 to 10 microns in diameter (Hyde). 

Trichophyton Megalosporon Endothrix. — This organism is 
the cause of tinea circinata (herpes tonsurans, ringworm of the 
body) and of tinea sycosis (tinea barbae, ringworm of the beard, 
barbers' itch). 

The trichophyton is composed of spores, which vary greatly 




Fig. 44.— Achorion Schonleini. 



in size, but which, as a rule, are somewhat larger than those of 
the type next to be discussed. They are frequently cuboidal, 
oval, or irregularly rounded, but their chief characteristic lies in 
their arrangement in lines or chains, extending up and down the 
hair shaft (see Fig. 45). The mycelium is found without, but 
never within, the hairs (Hyde). 

These fungi may be stained by the method of Morris and 
Calhoun. The hair is first washed in ether to remove all fatty 
debris; it is then put for one or two minutes in Gram's iodine 
solution and is stained after drying in gentian- violet for one to 
five minutes. It is again dried and treated for a minute or 
two with the iodine solution and for an equal length of time in 



VEGETABLE PARASITES. 



199 



aniline oil containing pure iodine, after which it is cleared with 
aniline oil, washed in xylol, and mounted in Canada balsam. 

Microsporon Audouini (Trichophyton Microsporon). — This 
parasite appears under the microscope chiefly in the form of a 
large number of round spores, irregularly grouped or massed 
about the follicular portions of the hair. Mycelial threads, large 
and branching, are often seen within the hair. The sheath of 
spores surrounding the hair is often continued upward for %6 
to Ys inch above its exit from the follicle and may be recognized 
as a whitish or grayish coating of the hair. These mycelial 
threads are all within the hair proper, thus differing from those 




Fig. 45.— Trichophyton Spores and Threads, 

of the trichophyton, which are never within the hair; after re- 
peatedly dividing and subdividing, they terminate on the outer 
surface of the shaft in fine filaments, at the extremities of which 
are the spores. This parasite is the cause of tinea tonsurans, or 
ringworm of the scalp. 

Microsporon Furfur. — This parasite is readily recognized by 
the microscopic examination of the scales scraped from the skin. 
Innumerable clustered spores, highly refractive and resembling 
in their circular and oval contours droplets of oil, are quite 
characteristic. The mycelial threads are not usually branched, 
but lie in a close network among which sporophores are dis- 
tinguishable, with conidia and terminal elements emerging at one 
extremity of the spore case. Both elements of this organism are 
more readily stained by the aniline dyes than are those of the 
trichophyton or favus. This organism is the cause of the condi- 
tion known as tinea versicolor. 



VIII. 

DETERMINATION OF THE FUNCTIONS 
OF THE STOMACH. 

THE VOMITUS. 

It is not good practice to utilize vomitus for the purpose 
of chemical analysis. Such material is usually of very uncertain 
composition, being contaminated with mucus from the upper 
part of the tract; further, the amount or composition of the 
food previously ingested is an unknown factor, affecting mate- 
rially the quantitative and qualitative findings. However, it 
may be found of decided advantage to test all vomited matter 
for the presence or absence of acidity and free hydrochloric acid. 
In the presence of suspected cancer the vomited material may 
be searched for necrotic tissue shreds or sarcinse. 

GASTRIC CONTENTS. 

The term gastric contents is a misnomer and should not be 
used in connection with chemical laboratory procedures. There 
is no gastric contents which can be pointed to as normal. 
There is always something in the stomach which may by proper 
methods be removed for examination, even from the fasting 
stomach. In the study of the gastric functions, samples are re- 
moved at certain times, either with or without preliminary ad- 
ministration of a test diet, for purposes of chemical and micro- 
scopic examination. The results of this examination are then 
compared with the normal findings, obtained under the same 
conditions in normal individuals. 

The recent work of Eehfus and others has done much to 
modify our earlier conceptions of the chemical activities of the 
stomach, so that at the present time the older method of ad- 
ministering a test-meal and then at any given time removing it 
for purposes of examination has little to recommend itself to 
the progressive physician. However, it seems best to allow this 
clumsy and inefficient method to remain, in this edition, with 
(300) 



GASTRIC CONTENTS. 



201 



the warning that it should never be employed when the more 
scientific methods are available. 

Fractional Method of Gastric Analysis. — The old method of 
test-meal extraction with a large tube, at the end of a definite 
period is very rapidly being superseded by the so-called ^'frac- 
tional method." This is far less distasteful to the patient be- 
cause of the small caliber of the tube used, which permits of its 
remaining in situ for hours if desired, during which time at 
stated intervals, small quantities of gastric contents may be 
removed for examination. By this method the observer is able 
to follow the complete cycle of gastric digestion. The use of 



Tot. 
Ac. 




A 


\ 




/- 




■■ w 


4^ 




N/10 
NaOH 


/ 


f 


X 


\^ 


^ 


"-^ 


-4- 


60 

30 


/^ 


X 


—5 


^\ 


«->„.,> 




\ 




Time 






Ihr. 






2hr. 



Fig, 46.— Normal and Pathologic Curves after an Ewald Meal, 
1. Normal Curve. 2. Delayed Digestion with Late Hyperacidity, 3. 
Larval-hyperacidity. 4, Tardy Hyperacidity. 5, Marked Continued 
Secretion from Obstruction (Hawk). 

this method has demonstrated the unreliability of the ''^one ex- 
traction" method, which often leads to gross misinterpretation 
and serious errors in diagnosis (Fig. 48). 

Technic. Method of Ebheus. — The Eehfus tube (Fig. 
47) is easily swallowed by the patient and when it has passed 
in to the desired length, the upper end is brought to the 
corner of the mouth and held in position by a piece of adhesive 
plaster attaching it to the cheek. The position of the tube in 
the stomach may be determined by aspirating some of the 
contents. 

Removal of Residuum. — ^Examination of the fasting stom- 
ach in tlie morning will show considerable material, all of 



202 DETERMINATION OF FUNCTIONS OF STOMACH. 

which, should be removed before the test-meal is introduced. 
If this is not done samples drawn after the test-meal cannot be 
considered representative of gastric secretory activity resulting 
from the stimulation of the test-meal. A residuum up to 50.0 
cubic centimeters is considered normal. It will be found to 
possess all the characteristics of physiologically active gastric 
juice^ with an average total acidity of 30 and a free HCl con- 
tent of 18.5. This fluid is often discolored with bile^ especially 
in fluids of relatively high acidity. Tests for trypsin are gen- 
erally positive. A large volume of residuum, of total acidity of 




Fig. 47.— Rehfdss Tube and Extraction Syringe. 

70 or over, strongly favors ulcer (Fig. 46) . According to Hawk, 
plain water has the same effect in provoking gastric secretory 
activity as the Ewald test-meal, so if desired this may be sub- 
stituted. The use of water has the additional advantage of per- 
m.itting the demonstration of food retention and to test more 
accurately for lactic acid, bile and blood. 

The Retention Meal. — This is composed of undigestible 
substances easily recognizable in the gastric residue which are 
added to the usual evening meal. Choice may be made from 
the following: Figs, dates, prunes or string beans. 

Removal of Samples for Analysis. — At intervals of exactly 
fifteen minutes from the time the test-meal is eaten until the 
stomach is empty, 5 to 6 cubic centimeter samples of gastric 
contents are withdrawn through the tubes by means of the glass 
syringe (Fig. 47). Care should be exercised not to exert too 



GASTRIC CONTENTS. 



203 



much suction. To completely empty the stomach aspiration is 
practised in four positions — (a) on the back, (&) on the abdo- 
men, (c) on the right side, (^), on the left side. This if care- 
fully done should result in complete evacuation of the organ. 
Three tests may be employed to determine this fact. (1) No 
more material can be aspirated in any position; (2) injection 
of air and auscultation while shaking over the stomach fails to 



100 



60 



60 



40 



20 



total ac 
free ac' 



:-v 




20 40 60 80 100 120 minutes 

Fig. 48.— Acidity Curves op Normal Human Stomach (Hawk). 



reveal splash-sounds; (3) lavage and aspiration through the 
tube will fail to return any food or other gastric material. 

Examination of Samples. — Strain each sample separately 
and examine the residue microscopically for mucus, blood-cells 
and food-rests. The strained contents is then examined for 
total acidity, free HCl, peptic activity and occult blood. 

Total Acidity. — ^Method of Serial Titratioi^: Arrange 
a series of numbered evaporating dishes corresponding to the 
number of samples to be titrated, introduce 1 cubic centimeter of 
the proper sample into each, dilute with 10 cubic centimeters of 
water and add 2 drops of phenolphthalein indicator, then add 
N/100 NaOH to the contents of dish No. 1 at a definite rate 



204 



DETERMINATION OF FUNCTIONS OF STOMACH. 



from a burette^ -antil a faint pink color throngliout the fluid in 
the dish is obtained. Eetnrn dish No. 1 to its place in the 
series and place dish No. 2 under the burette. Take burette 
reading for No. 1 and then titrate No. 2 in the same way. 
Continue throughout the series. 

Calcuiation". — Note the number of cubic centimeters of 
N/100 NaOH required to neutralize 1 cubic centimeter of 
stomach contents, multiply by 10 to obtain the number of cubic 



100 

X! 

o 

to 

z 80 

o 



60 



40 



20 



f^ 



t:^ 



"m 



t: 



±± 



m 



m 



'A 



Vz »/4 1 



m 



^SSffigJi]^: 



Total adctit^ 
Free acidity 



Ya V/z 1 2/4 2 hours 



Fig. 49.— Acidity Curves from Case of Hyperacidity (Hawk). 



centimeters of N/10, NaOHl required to neutralize 1 cubic 
centimeter of stomach contents and this figure multiplied by 100 
gives the number of cubic centimeters of N/10 NaOH required 
to neutralize 100 cubic centimeters of gastric contents. This 
is the method of calculation now usually employed, the results 
of which should be plotted in a form similar to that shown. 
(Fig. 48.) 

Curves Obtained by the Fractional Method. — Accord- 
ing to Hawk, a normal stomach after an Ewald test-meal will 
give a curve similar to that in the chart. The curve may vary 
within certain limits but will conform to the illustration. The 



GASTRIC CONTENTS. 



205 



meal has usually entirely passed out of the stomach at the end of 
two and one-half hours. Pathologically every variation may 
occur, both as to time of emptying and the character of the 
curve and the quantity of material found. Typical curves from 
cases of hyperacidity, gastric carcinoma and achylia, are taken 
from Hawk and shown in figures 49, 50 and 51. 

Deteemination" op Free Acidity. — The usual indicator 
employed is Topfer's dimethylamidoazobenzene. This may be 



1 

1120 
960 
800 „ 
640 o 

Z 

480 1 

320 80 

60 

160 40 






















/ 
/ 




Ga. 
Cai 


cinon 


a 




/ 
1 










•^/ 


1 










1 


. 










/ 












/ 

/ 








y 


y 
y 




Tot 


ilac 


^ 




^^.^ 





^ 




,^'' 




Frc 


iac 



Fig. 50. 



-Acidity and Protein Curves in Gastric Carcinoma 
(Clarke and Rehfuss). 



inaccurate because of an uncertain end-point. For this reason 
Sahli^s reagent (a mixture of equal parts of a 48 per cent, solu- 
tion of potassium iodide and an 8 per cent, solution of potas- 
sium iodate) is recommended as it gives values similar to 
Topfer^s reagent in average acidities (Hawk). 

Procedure. — Serial titrations are prepared as above des- 
cribed for total acidity, by measuring 1 cubic centimeter of 
strained gastric contents into several marked porcelain dishes. 
Each is diluted with 10 cubic centimeters of water and 1 cubic 
centimeter of Sahli's reagent added to each. Allow the stomach 
contents thus treated to stand for five minutes and then titrate 



206 



DETERMINATION OF FUNCTIONS OF STOMACH. 



with N/100 sodium thiosulphate, until only a faint yellow 
remains. Now add 5 to 10 drops of a 1 per cent, solution of 
soluble starch and continue the titration until the blue color 
disappears. 

Calculation". — Note the number of cubic centimeters of 
N/100 sodium thiosulphate required to titrate 1 cubic centim- 
eter of stomach contents to a total disappearance of blue color 
in the presence of starch. Inasmuch as N/100 thiosulphate is 
equivalent to N/100 alkali, this valu^ indicates the number of 
cubic centimeters of N/100 of NaOH necessary to neutralize the 
free HCl in 1 cubic centimeter of stomach contents. Multiply 
the value by 10, to obtain the number of cubic centimeters of 



80 
40 
















,..——•'*'* 











— • — 




Hours 


V4 Vz 3/4 1 IV4 \Vz 1%. 



Fig. 51.— Total Acidity and Protein Curves in Benign Achylia (Solid 
Line Represents Acidity). (Clarke and Rehfuss.) 

N/100 NaOH necessary to neutralize 100 cubic centimeters of 
stomach contents. Plot results in a curve similar to that shown 
in Fig. 48. 

Methods af Obtaining' Specimen for Examination. — The 
usual apparatus employed to remove the test-meal comprises the 
well-known gastric tube of soft red rubber, fitted at one end 
with a soft rubber funnel, and containing near this a bulbous 
expansion without valves. A recent modification of and im- 
provement over this is the large bulb devised by Ewald. This 
bulb is sufficiently large to contain the total quantity of material 
removed, thus overcoming in a measure the difficulties of the 
smaller bulb. These two methods are usually successful in 
obtaining the desired material for examination, but are difficult 
to manage and possess a decided disadvantage in that they 
do not provide for the important procedure of lavage and 
inflation. 



GASTRIC CONTENTS. 207 

Also, tubes of irregular caliber are difficult to cleanse, 
and unless great care is exercised in this direction, they may 
become a source of contamination, if not carriers of infection. 

Some years ago Dr. Judson Daland adopted for this pur- 
pose two large open-mouth bottles and a double-action Davidson 



Fig. 52.— Author s Apparatus for Gastric Test-meal Removal, 
lavage and inflation. 

bulb ; these are used in conjunction with the plain gastric tube. 
With this arrangement, aided by an assistant, it is possible to 
rapidly and cleanly obtain a sample of gastric contents (in con- 
siderably less than a minute), and to follow immediately with 
lavage and inflation if desired. 



208 DETERMINATION OF FUNCTIONS OF STOMACH. 

To those who have used this method with uniforin success 
in both private and hospital practice, its advantage is evident, 
the only drawback being the almost absolute necessity for trained 
assistance, this being occasioned by the complicated nature of 
the apparatus, which requires a second pair of hands to manipu- 
late the bottle and its connections during the passage of the tube. 
To overcome this drawback the author has devised a reversing 
valve (see Fig. 52). With this valve it is unnecessary to make 
any change in the tube connections when once the apparatus is 
set up. The perfected device has been worked out with great 
care, having in mind the necessity of a simple mechanism capa- 
ble of being cleansed and kept in repair without difficulty. 

PRELIMINARY PREPARATION OF THE PATIENT. 

The preliminaries leading up to the extraction of the test- 
meal should be as nearly uniform in every case as possible. By 
adopting a definite routine and adhering to it we eliminate, in 
a great measure, the errors which would otherwise creep in and 
lessen the value of the findings. The adoption of the following 
rules will enable the examiner to obtain a series of reports in one 
case or in groups of cases of far greater value than could be 
obtained by an irregular technic : — 

First. — It is advisable that all medication should be with- 
held until after the gastric analysis has been made. If this is 
impossible, then a period of days should elapse before making 
the test, during which all treatment is stopped. 

Second. — The test-meal should be given on an empty stom- 
ach, either one which has been emptied by fasting or by pre- 
vious lavage. 

Third. — The volume and composition of the test-meal 
should be uniform. The particular meal employed to be deter- 
mined by the operator according to his preference. 

Fourth. — 'The removal of the meal should (depending on 
its composition) be accurately timed; this time to be measured 
from the beginning and not from the end of the meal. 

Fifth. — The test-meal should be removed without dilution 
if possible. If dilution should be necessary to accomplish re- 
moval, the amount of water used should be definitely known. 



COMPOSITION OF USUAL TEST-MEALS. 209 

and the total withdrawn must exceed the amount used. If this 
rule is not observed, all investigations of a quantitative nature 
are valueless. For method of determining total volume of gas- 
tric contents, see page 215. 

Sixth. — The tube should remain within the esophagus only 
long enough to allow time to compress the bulb. If much time 
elapses after introduction before removal of the contents, the 
hypersecretion of mucus occasioned by the presence of the tube 
will alter the composition of the sample and possibly result in 
erroneous conclusions. 

A tube of relatively large diameter is to be preferred, being 
easier of insertion, less uncomfortable to the patient, and in- 
suring greater success in obtaining a sample. A suitable tube 
should measure between % and % inch outside diameter, and 
should be provided with both a terminal and lateral opening in 
the gastric end. 

A lubricant for the tip of the tube is unnecessary, the 
contact of the tube with the patient's pharynx immediately ex- 
citing sufficient flow of mucus for this purpose. This is prefer- 
able to either nauseating oil or hygroscopic glycerin. In case 
of great reflex excitability of the pharyngeal constrictors, this 
region may be first sprayed with a dilute solution of cocaine. 

COMPOSITION OF THE USUAL TEST-MEALS. 

(a) Ewald Test-meal. — This meal is, perhaps, the most fre- 
quently employed for general work. It consists of a roll, or 
piece of bread or toast (about 35 grams) without butter, and 2 
cups of water or tea (about 300 cubic centimeters) without milk 
or sugar. 1 

The contents are removed one hour later. This amounts 
normally to 30 to 50 cubic centimeters, depending both upon the 
skill of the operator and upon the condition of the stomach. 
Hypermotility of the stomach will diminish the quantity of con- 
tents recovered, while a hypomotility will increase the quantity. 

(&) Modified Ewald Breakfast. — Of the test-meals em- 
ployed, one of generally useful composition includes the white 

1 It has been shown that It is almost impossible to detect blood in the 
presence of tannic and gallic acids, so that it is advisable not to use tea in 
the test-meal when blood is suspected. 

14 



210 DETERMINATION OF FUNCTIONS OF STOMACH. 

of 2 eggs poached or soft boiled, without yelks and without 
seasoning; 2 pieces of toast without butter (the slightest trace of 
butter or fat will cause lactic and butyric acid fermentation), 
and a cup of tea without milk or sugar. 

This meal should be removed at the expiration of one hour, 
when, under ordinary circumstances, there will be recovered be- 
tween 30 and 90 cubic centimeters. 

(c) Boas Test-meal. — This meal consists of a dish of oat- 
meal prepared by concentrating to 500 cubic centimeters a liter 
of water to which a tablespoonful of oatmeal is added. The 
chief advantage of this meal is that it introduces a digestible 
material that is free from lactic acid, which is a normal con- 
stituent of bread. The contents of the stomach are withdrawn 
one hour later when, owing to the small volume of material in- 
troduced, the amount may be very small. If the stomach shows 
normal digestive powers most of the material will be then passed 
into the intestines, while an appreciable amount of material 
recovered would indicate either a dilatation of the stomach or 
pyloric obstruction. 

(d) Riegel Test-meal. — ^This test-meal has the advantage 
of permitting the patient to use a more normal diet than in the 
ones previously mentioned. This is of considerable importance 
in America, where we are not accustomed to the Continental 
breakfasts. 

The Eiegel meal is given in the middle of the day and con- 
sists of about 400 cubic centimeters of soup, 200 grams of beef- 
steak, and either 2 slices of white bread or 150 grams of mashed 
potato, together with one glass of water. This meal should be 
withdrawn at the expiration of three to four hours. The advan- 
tages of this meal are that they incite a more nearly normal 
gastric juice than the preceding ones, and give some indication 
of the rate and amount of the digestive process, but has the dis- 
advantage that particles of undigested food frequently clog the 
tube and interfere with the removal of the contents. 

(e) Fischer's Test-meal. — This meal, introduced by an 
American physician, has the advantage of more nearly approach- 
ing an American breakfast than the others. It consists of the 
bread and tea of the Ewald meal together with i/4 pound of finely 
chopped lean beef broiled and seasoned. The contents are re- 



DALAND-FAUGHT TEST-MEAL APPARATUS. 211 

moved at the end of three hours. Fischer has shown, by com- 
paring results after this meal with those of the Ewald breakfast, 
that with his combination the results are much more constant, 
though somewhat higher, than with the other Ewald or Boas 
brealdasts. 

(/) Salzer Test-meal. — This is a double meal and is given 
as follows : For breakfast the patient receives 30 grams of lean 
cold roast meat, finely chopped; 250 cubic centimeters of milk; 
60 grams of rice, and 1 soft-boiled egg. Four hours thereafter 
a second meal is given, consisting of 37 to 70 grams of stale 
wheat bread and 400 cubic centimeters of water. The contents 
are removed one hour after this second meal. Under normal 
conditions of digestion and motility the stomach contents should 
show no renmants of the first meal. 

THE DALAND-FAUGHT TEST-MEAL APPARATUS. 

The component parts of the apparatus are as follows : — 

A plain gastric tube without bulb or funnel. 

A double-action Davidson hand-bulb. 

A large rubber stopper having two perforations. 

Two wide-mouth bottles containing more than a liter each, 
and graduated in cubic centimeters.^ 

A short length of large glass-tubing and some i/t i^^h 
rubber-tubing. 

The reversing valve. 

Technic of Removal. — Fill one graduated bottle to the 500 
cubic centimeter mark with warm sterile water. Fit the double 
perforated stopper with the glass tube and the reversing valve. 
Place the stopper firmly in the empty bottle and attach the gas- 
tric tube to the glass tube, and the two ends of the double action 
bulb to the tw^o horizontal tubes of the valve (Fig. 52) . Finally, 
ascertain the direction of the air-current through the valve by 
making a few pressures on the bulb. Set the valve to make 
negative pressure within the bottle. 

The tube should now be passed, with the patient preferably 
in the sitting posture. As soon as the tube enters the cardiac 

2 These special bottles are not necessary for practical purposes; any large, 
open mouth bottle of sufficient quantity, such as a quart milk bottle, may be 
substituted. A mark must be made at the measured 500 cubic centimeter mark 
on one bottle. 



212 DETERMINATION OF FUNCTIONS OF STOMACH. 

orifice, a few quick pressures are made on the bulb. This de- 
velops a slight degree of negative pressure within the bottle, 
when the gastric contents will immediately flow into the bottle. 
Sudden stopping of the flow from occlusion of the tube by par- 
ticles of food or mucus may immediately be removed by mo- 
mentarily reversing the lever. This will cause a small portion 
of the gastric contents to return through the tube, effectu- . 
ally washing out the obstruction. This simple maneuver may 
be repeated as frequently as necessary to obtain a sufficient 
specimen. 

In the event of failure to obtain sufficient material by this 
means, the difficulty will usually be overcome by the introduc- 
tion of a measured amount of water. To accomplish this the 
stopper with all its connections is removed from the bottle and 
fitted into the bottl-e containing the 500 cubic centimeters of 
warm water. The valve is set to make positive pressure within 
the bottle, and by means of the bulb about 400 cubic centimeters 
are run into the stomach; then by reversing the valve the whole 
is withdrawn. It is necessary to recover more than a total of 
500 cubic centimeters if any determinations of a quantitative 
nature are to be made. 

If it has been necessary to resort to the introduction of 
water to effect the removal of the test-meal, allowance must then 
be made in the final calculations, so that the results will repre- 
sent the quantities in pure gastric contents. 

For Example: Suppose, after employing 500 cubic centi- 
meters, a total of 550 cubic centimeters is recovered; of this 
amount only 50 cu.bic centimeters represent actual gastric con- 
tents, or 1 part in every 11. It will be necessary then to mul- 
tiply any figures obtained in the calculations of acidity by the 
factor 11 in order to express the results in terms of undiluted 
gastric contents. 

Tiirck's Double Stomach-tube. — F. B. Tiirck^ has devised a 
double tube consisting of two single tubes placed parallel to each 
other; the smaller, inlet tube, has a diameter of 5 millimeters; 
the larger, outlet tube, has a diameter of 12 millimeters. The 
outlet tube is longer by 10 centimeters than the inlet tube, 
thereby allowing the larger tube to reach the floor of the stomach. 

3 Chicago Medical Record, December, 1907. 



GASTRIC CONTOUR AND POSITION. 213 

The contour of the two tubes fused together is that of a 
flattened oval, which conforms to the shape of the esophagus. 
This tube is said to be more flexible and therefore less uncom- 
fortable to the patient than the regular tube and at the same time 
allows of a maximum outlet for the stomach contents and, be- 
cause of the pressure exerted by the bulb, assures a maximum 
of successful removals. 

Method of Use. — After the tube has been inserted to the 
proper distance, the large inflating bulb is placed on the upper 
end of the inlet tube. A sudden puff of air from the bulb will 
insure immediate expulsion of the stomach contents through the 
outlet tube. This apparatus may also be used for purposes of 
determining the gastric contour by inflation. 

TO DETERMINE GASTRIC CONTOUR 
AND POSITION. 

Inflation of the Stomach. — Next to the X-ray, probably the 
best method of determining the size, shape, and location of the 
stomach is by the introduction of air. To accomplish this two 
methods are available. Of these, the second is greatly to be 
preferred for reasons to be stated later : — 

1. The flrst consists in administering 1 dram of sodium 
bicarbonate dissolved in a little water, to be immediately fol- 
lowed by an equal quantity of tartaric acid, also in solution. 
The combination of these causes the evolution of carbon dioxide 
gas within the stomach, which immediately distends that organ. 
This method is open to serious objection, because the quantity 
of gas produced cannot be controlled and overproduction, be- 
sides causing great discomfort if not doing actual damage, may 
result in hemorrhage and great cardiac embarrassment. On 
the other hand, sufficient gas may not be evolved to completely 
distend the stomach, and thus its full size and shape fail to be 
accurately determined. In the light of these facts it would 
seem best that this method be permanently abandoned for the 
following, which is more in accord with the principles of scien- 
tific medicire: — 

3. The second method of inflation is accomplished throiigli 
the stomach-tube by means of a Davidson bulb. This simple 
combination may be employed with safety and accuracy, and 



214 DETERMINATION OF FUNCTIONS OF STOIiIACH. 

even in the absence of the graduated bottles and reversing valve 
can be made to serve. 

A greater refinement in the technic is attainable with the 
aid of bottles and valve above described. Their use may con- 
venientl}^ follow the removal of the test-meal. With this outfit 
in addition to inflation we ma}^ also roughly measure the cubic 
contents of the distended stomach, by introducing a measured 
quantity of air. 

Technic of Inflation. — By inflation the position of the stom- 
ach may be accurately outlined. After the test-meal has been 
removed the patient is placed in the semi-recumbent posture, 
and the empty stomach is outlined as accurately as possible by 
auscultatory percussion. Air is then introduced into the stomach 
through the tube in suflicient quantity to produce a distinct 
change in the auscultatory percussion-note. The quantity of air 
required to accomplish this is quite small, not sufficient to alter 
the relation of the organ to surrounding viscera, as is the case 
when the stomach is ballooned with air. By the proper working 
of the valve and the bulb on the apparatus, we can change the 
gastric note at will, thereby being able to differentiate abso- 
lutely between gastric and colonic tympany. 

By this method gastroptosis can be absolutely determined, 
the author having in many instances had the results confirmed 
by the X-ray. 

Contraindications to Inflation. — These are the same as 
for the use of the stomach-tube itself, viz. : Myocardial degen- 
eration, with or without endocarditis; angina pectoris, aneurism, 
advanced vascular degeneration, hemorrhage from any part of 
the body, and all diseases in which hemorrhage is likely to occur. 
Gastric ulcer is, of course, a contraindication, especially when 
hemorrhage has been noted. Another contraindication, and one 
which is not sufficiently emphasized, is neurasthenia and allied 
mental states with gastric symptoms. In these diseases tlie 
symptoms referable to the stomach are not due to organic change 
in that organ, but are psychic in origin. The use of the tube 
in these cases serves but to reinforce the idea of disease of the 
stomach, and renders a cure more difficult and occasionally im- 
possible. This, of course, refers to those cases which are ob- 
viously psychic in origin. In certain cases it is not possible to 



GASTRIC CONTOUR AND POSITION. 



215 



determine whether the disease is organic or not; here the tube 
would have to be used for diagnostic purposes. It has been sug- 
gested that the unpleasantness of the passage of the tube itself 
will act as a curative agent. Experience teaches that usually 
more harm than good will come from the use of the tube with 
this intent. 

To Determine the Capacity of the Stomach by Inflation. — 
To accomplis]] this the apparatus employed in the preceding de- 




c.c 



_l( 00 

-J_ JO 

"j-to 



c.c. 



Fig. 



53.— Diagrammatic Representation of Arrangement of Bottles 
FOR Measuring Cubic Contents of Stomach. 



scription must be augmented by the addition of a second double- 
perforated rubber stopper and a second section of glass tubing, 
arranged in a similar manner to the first. 

The apparatus is to be set up as follows (see Fig. 53) : The 
extremity of the gastric tube is attached to the short glass tube 
of the second stopper, and a section of tubing made to join the 
long glass tube of each stopper. The bottle belonging to the 
valve and bulb is filled with water to the 1000 cubic centimeter 
mark, and both stoppers placed in their respective bottles. Now, 
with the tube in the stomach and the valve set for compression, 
the water is gradually forced over into the second bottle. This 
in turn displaces the air, and forces it into the stomach. AYhen 



216 DETERMINATION OF FUNCTIONS OF STOMACH. 

the patient indicates that the stomach is full, the amount of 
water displaced from the first bottle will equal the amount of 
air forced into the stomach, and can be read in cubic centimeters 
from the scale on the bottle. (See diagram.) The valve should 
now be reversed and the air withdrawn. 

Technic of Gastric Lavage. — Prepare a large pitcher of 
sterile water at body temperature, and a receptacle for waste. 
(If desired normal saline or dilute alkaline water may be sub- 
stituted.) Place 500 cubic centimeters of the wash-solution in 
one bottle, and by means of the valve and bulb force a few hun- 
dred cubic centimeters into the stomach; then, after allowing it 
to remain for a few moments, reverse the valve and withdraw as 
much as possible. Discard the returned water and repeat this 
process until the wash-water returns clear. A quantity of this 
saline or alkaline solution may be allowed to remain when the 
tube is finally withdrawn. 

Indication for the Use of the Gastric Tube. — The tube is 
positively indicated in cases of carcinoma associated with py- 
loric stenosis. The result here is decided relief from distressing 
symptoms, and often actual prolongation of life. 

In certain cases of pernicious anemia, especially in the later 
stages, stagnation of food gives rise to distressing symptoms, and 
by allowing absorption of toxins hastens the progress of the 
disease. 

Also, in cases where poison has been taken into the stom- 
ach in toxic doses, the stomach tube may be of great service in 
affording prompt removal. 

Contraindications to passing the tube are : uncompensated 
heart disease of any kind, either muscular or valvular ; aneurisms, 
advanced pulmonary tuberculosis, apoplexy, or recent and very 
severe hemorrhage from the stomach, especially in the case of 
ulcer or carcinoma of that organ. Menstruating and preg- 
nant women should not be examined unless the diagnostic or 
therapeutic results expected are of great importance. Ulcer of 
the stomach is not an absolute contraindication, however, if 
great gentleness is used in passing the tube, and the stomach is 
not too much distended by fiuid. The tube should be used only 
for important diagnostic or therapeutic purposes. It is safer to 
treat a suspected case as ulcer ^ind avoid the passage of the tube. 



SECRETION OF HYDROCHLORIC ACID. 217 

THE CHEMISTRY OF DIGESTION. 

GASTRIC ACIDS AND ACIDITY. 

Gastric Contents from Fasting Stomach. — The stomach is 
practically never empty, but always contains a certain amount of 
acid fluid. Boas considers anything between 1 and 100 cubic 
centimeters as a normal amount of material for the fasting 
stomach. Anything above this amount would mean either motor 
insufficiency or hypersecretion. One may differentiate these two 
conditions by washing out the stomach at night, when the 
material withdrawn in the morning will be extremely scanty if 
the condition is one of motor insufflciency. (See page 201.) 

This fluid from the fasting stomach is thin, has a specific 
gravity of 1004 to 1005, contains some free hydrochloric acid, no 
lactic acid, and no bacteria. It is very commonly bile-stained, 
may be alkaline from the presence of pancreatic juice, and may 
contain large amounts of mucus. As such material is always 
found in the fasting stomach, it is well to make it a rule to wash 
out the stomach the night before giving a test-meal. 

CHEMICAL COMPOSITION OF THE GASTRIC JUICE 
(containing water with saliva). 

Water 994.40 parts. 

Solids 5.60 " 

Organic material 3.10 

Mineral salts 2.50 

Sodium chlorid 1.46 

Calcium chlorid 0.16 

Potassium chlorid 0.55 

Ammonium chlorid Trace. 

Calcium phosphate Trace. 

Magnesium phosphate Trace. 

Iron 0.12 

Free HCl 0.20 

SECRETION OF HYDROCHLORIC ACID. 

Under conditions of health, after from ten to fifteen min- 
utes following the ingestion of food the gastric contents are 
acid, due to the presence of free acids or acid salts. At this 
time the free acid recognized is lactic acid. Up to thirty-five 



218 DETERMINATION OF FUNCTIONS OF STOMACH. 

or forty minutes lactic acid predominates, and only traces of 
HCl can be detected. Shortly after this the lactic acid disap- 
pears and only HCl remains, so that at the end of one hour no 
lactic acid can be demonstrated. 

Hydrochloric acid is actually present from the beginning, 
but its presence is masked by the excess of lactic acid and the 
HCl combined with bases. Free HCl increases with the progress 
of digestion until it reaches 0.15 to 0.2 per cent, after a light 
meal, or from 0.2 to 0.33 per cent, after an abundant meal. 

QUALITATIVE TESTS. 

For the simple qualitative demonstration of free acids, 
organic and inorganic, the Congo red and tropeolin papers are 
convenient. The former turns dark blue, the latter dark brown, 
when moistened with a solution containing free acids, but neither 
of these react to acids which are combined with bases. 

Detection of Free Hydrochloric Acid. — For the qualitative 
determination of the presence of free HCl, a number of tests 
are available. 

Tests. — 1. Topfer's Dimethyl-amido-azobenzol : For this 
test either the 0.5 per cent, alcoholic solution of this chem- 
ical can be used, or for convenience filter-paper may be soaked 
in the 0.5 per cent, solution, allowed to dry, and then kept bot- 
tled for use. This solution is delicate for 0.003 per cent. HCl. 
Combined HCl as well as acid salts and inorganic acids, in the 
concentration in which they occur in the stomach, will not 
cause this solution or the prepared paper to become pink. A 
pink reaction denotes the presence of free hydrochloric acid. 

2. Gunzberg's Phloroglucin Vanillin (for reagent see 
Appendix). — This reagent, if active, is of a pale-yellow color. 
It darkens and deteriorates on exposure to light; so should be 
kept in a dark-colored bottle or prepared fresh each time that 
it is required. A drop or two of this reagent is placed in a 
porcelain dish, together with an equal amount of filtered gastric 
contents, and heat gently applied until the liquid has evapo- 
rated. If HCl be present a rose-red color will appear at the 
margin of the evaporating fluid. This test is unmistakable and 
is most delicate, demonstrating the presence of free HCl in the 



QUALITATIVE TESTS. 219 

proportion of 0.05 per 1000. The reaction is not interfered 
with by albuminates, by salts present in the usual amount, or by 
organic acids. 

3. BoAs's Test. — This reagent consists of 5 grams of resorcin 
and 3 grams of cane sugar dissolved in 100 grams of 95 per ceiU. 
alcohol. It has the same delicacy as Gunzberg's test and is more 
stable. The test is applied in the same way as the preceding, 
taking particular care to use a low flame in evaporating, and it 
gives a rose-red or vermilion color in the presence of mineral 
acids. This color gradually fades on cooling and is not given by 
organic acids or acid salts. 

4. Tropeolin Test. — The reagent for this test is a satu- 
rated alcoholic solution of tropeolin 00. This test is applied in 
the same way as the preceding and gives a lilac-blue color in the 
presence of free acid. This test is not as delicate as the pre- 
ceding, reacting only in the presence of 0.03 per cent, of free 
hydrochloric acid. The presence of lactic acid in strength of 
0.24 per cent, or more gives the reaction. 

5. Von den Yelden's Methyl Violet Test. — Add a few 
drops of a saturated aqueous solution of methyl violet to a test- 
tube three-fourths full of water. The dilute solution of the dye 
should be clear and of a violet or purple color. This dilute 
stain is divided equally between two test tubes. To one add 5 
to 10 cubic centimeters of filtered gastric juice ; to the other an 
equal quantity of water. The presence of free HCl will be 
shown by a change in color from violet to blue; the other tube 
by comparison serving as a control. The test is positive in the 
presence of 0.025 per cent, of free HCl. Eoughly, this test 
when positive shows sufficient HCl present for proteid digestion. 

Lactic acid in less volumes than 0.04 per cent does not 
interfere with the reaction. 

6. Friedrich Test. — Friedrich'* uses neither sound nor 
special apparatus. His method consists in producing the neces- 
sary chemical reaction directly in the stomach by means of 
threads saturated with Congo red. A small metal cylinder witli 
lounded edges is enclosed in a capsule and attached to a loiijz 
thread dyed with Congo red ; another dyed thread i? attached to 

4 Berlin, klin. Wochen., July 22, xlix. No. 30. 



220 DETERMINATION OF FUNCTIONS OF STOMACH. 

the cylinder with one end left free and enclosed within the cap- 
sule. This capsule, with its enclosed metal cylinder, is swallowed 
readily by the patient, twenty minutes after the usual test- 
breakfast has been given. Half an hour later, it is withdrawn 
by means of the long thread when, according to the degree of the 
reaction on the thread, he estimates the amount of free hydro- 
chloric acid. The author has had no difficulty in getting his 
patients to swallow this capsule and he has repeatedly verified 
his findings by means of other tests. It should be mentioned 
here that the long thread is d3^ed a deep red, whereas the short 
thread is dyed pink, so that the degree of reaction may be noted. 
The author has laid down the following scale of the reaction for 
himself; browning of the dark-red thread indicates subnormal 
acid; violet, normal; blue-black, above normal or hyperchlor- 
hydria. In the presence of hyperchlorhydria, the pink thread is 
colored blue if the condition is of high grade, and sky blue if 
the hyperchlorhydria is extremely intense. The reaction must be 
noted immediately when the thread is withdrawn and before it is 
touched with fingers or instruments. 

QUANTITATIVE ESTIMATION OF TOTAL ACIDITY. 

The reaction of the filtered gastric juice being determined 
by the Congo red or the tropeolin papers, the total acidity is next 
determined by titrating against a decinormal sodium hydrate 
solution. 

Technic. — A Mohr burette is filled to the "0" mark with 
standardized decinormal sodium hydrate solution. 

To 10 cubic centimeters of filtered gastric juice in a por- 
celain dish, 10 cubic centimeters of distilled water is added, and 
1 or 2 drops of a 1 per cent, alcoholic solution of phenol- 
phthalein added as an indicator. (This indicator is inactive in 
the presence of carbon dioxid.) In the presence of free acids 
this mixture is colorless. The sodium hydrate is now run in 
slowly with constant stirring, until the rose color, which appears 
on the addition of each drop of alkaline solution, no longer dis- 
appears nor is intensified by further addition of the sodium 
hydroxid solution. 

As a rule, the acidity of the gastric contents, one hour after 
the ingestion of the test-breakfast, requires from 4 to 6 cubic 



AMOUNT OF GASTRIC JUICE SECRETED. 221 

centimeters of decinormal KaOH to neutralize 10 cubic centi- 
meters of gastric contents. For example, suppose 5.2 cubic 
centimeters were required to neutralize 10 cubic centimeters of 
gastric contents (this is within normal limits). The result of 
the estimation is usually expressed in parts of decinormal NaOH 
per 100 parts of gastric filtrate. This expression is easily ob- 
tained by moving the decimal point one place to the right. The 
result in the above example would, therefore, be recorded as 52 
parts of decinormal NaOH per 100 parts of gastric contents. 

QUANTITATIVE ESTIMATION OF FREE 
HYDROCHLORIC ACID. 

To 10 cubic centimeters of filtered gastric juice add an equal 
quantity of distilled water, and 1 or 2 drops of Topfer's 
reagent. This mixture, placed in a porcelain dish, is titrated with 
a decinormal solution of NaOH until the pink color of the solu- 
tion has been entirely removed and is replaced by pale yellow. 
Suppose, for example, 6 cubic centimeters were the amount 
required to neutralize the free HCl contained in 10 cubic centi- 
meters of gastric contents. To obtain the percentage of HCl in 
the gastric contents the following calculations are required : — 

One cubic centimeter of decinormal NaOH solution is 
equivalent to 0,00365 gram, of HCl (decinormal NaOH is equiva- 
lent to 4 grams of NaOH dissolved in exactly 1000 cubic 
centimeters of distilled water; each 1 cubic centimeter of this 
solution should exactly neutralize 0.00365 gram HCl). There- 
fore, 6 x 10 would equal the number of cubic centimeters of 
NaOH solution required for every 100 cubic centimeters of 
gastric contents, and the result, 60 x 0.00365, would equal the 
percentage of HCl in the specimen under examination, which 
would be 0.219 per cent. HCl. 

DETERMINATION OF THE TOTAL AMOUNT OF 
GASTRIC JUICE SECRETED. 

In order to determine the total amount of gastric juice 
secreted, a somewhat complicated procedure is necessary. It is 
evident that measurement of the quantity removed does not give 
us the total secretion, as it is impossible to obtain the last few 
cubic centimeters from the stomach. 



222 DETERMINATION OF FUNCTIONS OF STOMACH. 

Biy the method of Mathieu and Eemons as much of the gas- 
tric contents as possible is withdrawn into one graduated bottle 
and set aside. A measured quantity (300 or 400 cubic centi- 
meters) of water is then run into the stomach from the second 
bottle, allowed to remain for a few minutes, and is then with- 
drawn into the second bottle. If h is the quantity of gastric 
juice obtained by direct removal before the addition of water, a 
the total acidity of this undiluted juice, c the acidity of the 
diluted gastric juice, and ^ the amount of water added to the 
stomach, then the acidities a and c are inversely as the quantity 
of water used, since the greater the amount of the wash-water 
the less the total acidity of the second fluid withdrawn. 
This may be expressed by the following formula : — 
a: c = q -\- X'.x 
cq 

From which we derive x = 

a — c 
The amount of gastric juice originally present in the stom- 
cq 

ach is therefore h -) 

a — c 

This formula assumes that acid is present in the stomach 
contents. In diseases where no free acid is present, sufficient 
-^ acid may be given by the tube to cause an acid reaction in the 
stomach before proceeding, by which the combined acid may be 
estimated. 

Causes of Lowered Gastric Secretion.— I. In acute and 
chronic gastric inflammation. 

II. Atrophy of the gastric mucosa from any cause. 

III. General functional depression. 

IV. Expression of a gastric neurosis. 
v. Congenital idiosyncrasy. 

DETERMINATION OF ORGANIC ACIDS. 

These include lactic and acetic, and the true fatty acids, 
particularly butyric. Acetic and fatty acids are not found dur- 
ing normal digestion, and if present, as they sometimes are, have 
either been introduced with the food or have been produced by 



DETERMINATION OF ORGANIC ACIDS. 223 

fermentation of carbohydrates set up in the stomach by bacteria 
introduced with the saliva. 

The physiologic presence of lactic acid during the first stage 
of gastric digestion may be due either to its formation within 
the stomach, or from its having been introduced with the food, 
as in baked bread. 

Uffleman's Test for LaCtic Acid. — The addition of a 
few drops of filtered gastric contents to TJffleman's reagent (see 
Appendix) in a test-tube will, in the presence of lactic acid, 
change the amethyst-blue to a canary-yellow. This test is posi- 
tive in the presence of 1 part of lactic acid in 20,000. 




Fig. 54.— Strauss's Separatory Funnel. 

Sources of Error. — Lactates cause the same reaction. This 
is, however, immaterial, since we desire to recognize lactic acid, 
whether free or combined. The reaction also takes place with 
alcohol, sugar, and certain salts, particularly the phosphates, but 
rarely in their usual concentration after the test-breakfast. 

Steauss's Method for Lactic Acid. — This method is, per- 
haps, the very best clinical method at our disposal, as it shows 
lactic acid when present in pathologic amounts. It does not, 
however, give a quantitative result, nor does this seem necessary 
in the ordinary clinical work. Into a special separatory funnel 
(see Fig. 54) are introduced 5 cubic centimeters of the gastric 
Juice. The funnel is then filled to the 25 cubic centimeter 
mark with alcohol-free ether and well shaken. The ethereal 
layer will take up the lactic acid from the gastric contents. After 
the fluids have settled, open the stop-cock and run off the mix- 
ture down to the mark 5, after which distilled water is added to 
make up the 25 cubic centimeter volume. Two drops of 10 per 



224 DETEEMINATION OF FUNCTIONS OF STOMACH. 

cent, ferric chlorid solution are then added A\^ith a medicine 
dropper and the mixture well shaken. The water will now 
extract the lactic acid from the ether. The aqueous layer is 
colored an intense greenish yellow if more than 0.1 per cent, of 
lactic acid is present^ while smaller amounts will show a slight 
greenish tinge. This test may be negative if the lactic acid 
present is completely combined with the proteins of the gastric 
juice. In such cases hydrochloric acid may be added to liberate 
this lactic acid before shaking out with ether. 

Kelling's Test for Lactic Acid. — Dilute a small portion 
of gastric filtrate mth-10 to 20 parts of distilled water, in a 
test-tube. Fill a second tube with the same quantity of distilled 
water. To each tube add 1 drop of standard ferric chlorid 
solution. The presence of lactic acid in the test solution will 
be shown by the appearance of a canary yellow color. 

Lactic acid in the strength of 1 : 12,000 may give a positive 
reaction. 

The fatty acids, particularly butyric, strike a tawny-yellow 
color with a reddish tinge with Uffleman's reagent. The reac- 
tion is positive with 1 part in 2000. Fatty acids may also be 
detected by heating to boiling a few cubic centimeters of gastric 
filtrate in a test-tube over the mouth of which a strip of moist 
neutral litmus paper is placed. On this the vaporized volatile 
acid will produce the usual change. 

Butyric Acid (Pineapple Test). — If a portion of the dried 
ethereal extract of the gastric juice (see method of Strauss 
above) be treated with a few drops of concentrated sulphuric 
acid and a little alcohol, the odor of ethyl butyrate is perceptible 
on slight warming. This odor is the peculiar one of pineapples 
and is very easily recognized. 

Acetic acid is easily recognized by its odor, but it may 
also be detected by neutralizing a watery residue after the re- 
moval of an ethereal extract, with sodium carbonate, and then 
adding neutral ferric chlorid solution. In the presence of 
acetic acid a striking blood-red color will be produced. This 
neutralization of the aqueous solution is an essential point in 
this test, as the presence of free acid will prevent the appearance 
of any precipitate, and the presence of free alkali will cause the 
formation of ferric hydroxid, which will mislead, as the colora- 



MICROSCOPIC EXAMINATION OF GASTRIC CONTENTS. 225 

tion is very much the same. A similar reaction occurs in the 
presence of formic acid, but this acid is never present in gastric 
juice. 

Alcohol. — This is sometimes formed in the stomach during 
intense yeast fermentation. This may be detected by the Lie- 
hen's iodoform test, applied as follows: To a portion of the 
gastric filtrate add a small portion of liquor potassii, and then 
a few drops of a solution of iodine and potassium iodide in water. 
(See Appendix.) If alcohol be present a yellowish scum gradu- 
ally occurs on the surface, which is readily recognized as iodo- 
form by its odor. The same reaction occurs in the presence of 
acetone, but occurs more rapidly. 

Propeptone and Peptone. — These are products of albumin 
digestion, and when present indicate the activity of this part 
of the gastric function. A few drops of filtrate added to hot 
Fehling^s solution produce a purplish color, if they are present. 

Starch. — ^The addition of a drop of LugoFs solution to a 
piece of filter paper upon which some unfiltered gastric contents 
has been allowed to fall will, in the presence of starch, yield a 
blue reaction. This reaction is intensified by the addition of a 
drop of crude nitric acid. 

MICROSCOPIC EXAMINATION OF GASTRIC 
CONTENTS. 

The examination of the gastric contents would not be com- 
plete without an examination of the sediment, both in an un- 
stained wet preparation and a stained dry specimen. 

For this, a small portion of the unfiltered material should 
be placed upon a clean slide and covered by a large cover-glass ; 
and examined by the low and medium powers, when much con- 
firmatory information may be obtained. 

Under normal conditions, various budding forms of yeast 
cells will be seen; also food remains, the character of which will, 
of course, be determined by the nature of the test-meal and the 
presence or absence of gastric retention and fermentation. 
Starch cells will be recognized by their irregularly oval oyster- 
shell forms, with eccentric striations. Meat fibers and muscle 
cells will show the characteristic striations, which will become 
more indefinite as the process of peptic digestion progresses. 

15 



236 DETERMINATION OF FUNCTIONS OF STOMACH. 

Elastic fibers will have their characteristic fibrillated, wavy 
appearance. 

Vegetable and fruit cells have their own peculiar appear- 
ance, which will be easily recognized. Occasional epithelial 
cells are encountered, and granular indeterminate debris goes 
to make up the bulk of the solid material present. This contains 
a var}dng amount of non-pathologic bacteria. Stagnant or de- 
composed specimens will show a larger number of yeast cells and 
crystals of calcium oxalate, tyrosin, cholesterin, etc. 

In pathologic conditions red blood-cells and pus-cells may 
be recognized, and more rarely tissue fragments, which by ap- 




FiG. 55.— BOAS-QPPLER Bacillus in Gastric Contents. 

propriate methods of staining may be identified as portions of 
carcinoma or other tumor. Occasionally flagellates, amebas, and 
other monads may be seen. 

The specimen employed above may be used for staining. By 
sliding the cover-glass off, a very fair smear will be made, which 
after drying and fixing may be stained by any of the simple dyes. 

Examination of the stained specimen with the %2 objective 
will show innumerable micro-organisms, among which may be 
mentioned sarcince ventriculi, which are usually considered evi- 
dence of dilatation and fermentation, but which are not them- 
selves pathogenic. 

The Boas-Oppler Bacillus. — This organism is found quite 
commonly in patients suffering with carcinoma of the stomach, 
and rarely in non-malignant disease. It is found more fre- 
quently in the gastric contents at a time when lactic acid is 



STOMACH EXAMINATION FOR LEUKOCYTES. 227 

present in large amounts, so that in the incipient stages of car- 
cinoma these organisms may be absent. These bacilli are very 
large (3 to 10 microns by 1 micron) and are frequently joined 
end to end^ forming very long chains (see Fig. 55). They are 
readily stained with the usual aniline dyes and by Gram's 
method. On being treated with the dilute iodin, they take on a 
brown color, which distinguishes them from the large mouth 
bacillus {Leptothrix buccalis), which stains blue with iodine. 

SPECIAL EXAMINATION OF STOMACH 
FOR LEUKOCYTES. 

Eobertson and others have demonstrated the close relation 
of leukocytes in the washed stomach contents to gastric car- 
cinoma. This examination is made by first carrying out a thor- 
ough gastric lavage and then using the last clear water for the 
examination. A portion of this water is centrifuged and the 
sediment smeared on a slide, dried, and stained. The presence 
of leukocytes in this preparation is said to be almost conclusive 
evidence of gastric carcinoma. 

TESTS FOR OCCULT BLOOD. 

Blood may be intermittently present in the gastric contents 
or gastric vomitus as a result of one of the following conditions: 
I. Ulcer of the stomach or intestines. 
II. Benign pyloric stenosis. 

III. Spasm of the pylorus. 

Occult blood is usually constantly present in cases of 
malignant disease of the stomach or esophagus. 

Boas Modification of the Weber Test. — ^To 15 cubic 
centimeters of gastric contents, add an equal amount of ether 
and thoroughly agitate. To this mixture add 3 to 5 cubic centi- 
meters of strong glacial acetic acid, and again agitate. Now 
allow the mixture to settle and decant 10 or 15 cubic centimeters 
of the clear supernatant liquid, and to this add 4 or 5 cubic 
centimeters of ozonized oil of turpentine. In the presence of 
blood a violet or blue color will appear, which is intensified by 
the addition of chloroform. (See also test on page 255.) 

Acetic Acid Ether-Guaiac Test. — To 10 cubic centi- 
meters of gastric contents add 10 cubic centimeters of ether and 



228 DETERMINATION OF FUNCTIONS OF STOMACH. 

5 cubic centimeters of strong acetic acid. Thoroughly shake, 
and add 2 or 3 grains of powdered gum guaiac, and again 
agitate; allow to settle, and then add a few drops of fresh solu- 
tion of hydrogen dioxide. In the presence of blood a purple or 
blue ring will appear at the line of contact, or the solution grad- 
ually assumes a grayish-blue color. (See Chapter VIII, page 
258, for phenolphthalein test.) 

1. ORTHO-TOLiDii;r Keaction. — To 1 cubic centimeter of a 
4 per cent, glacial acetic acid solution of ortho-tolidin in a test- 
tube add 1 cubic centimeter of gastric residue or filtrate and 1 
cubic centimeter of 3 per cent, hydrogen peroxide. In the pres- 
ence of blood a bluish color sloAvly develops which may remain 
for several hours. 

2. Benzidin Reaction. — Add to 1 cubic centimeter of gas- 
tric material 3 to 5 cubic centimeters of benzidin solution (a 
saturated solution of benzidin in alcohol or acetic acid) and an 
equal volume of fresh 3 per cent, hydrogen peroxide. (If not 
already acid render so by the addition of acetic acid.) The 
appearance of a blue color denotes blood, control the test by 
substituting an equal volume of water for the gastric contents. 

ESTIMATION OF PEPTIC ACTIVITY. 

The digestive power of the filtered gastric contents from the 
recovered test-meal depends upon, first, the amount of pepsin 
contained, and, second, upon the amount of free acid present, 
particularly the free hydrochloric acid. Artificial digestion is 
the only means at our command by which we may determine the 
peptic activity of the gastric juice. 

Method of Ewald. — Prepare from the albumin of eggs, 
which have been boiled just sufficiently to cause firm coagula- 
tion, small discs or squares by first cutting thin slices of the 
coagulated albumin, and from these slices the cubes or discs. 
These bits of albumin may be prepared in bulk and preserved 
in glycerin, which is carefully washed off before using. 

The Test. — Place an equal quantity (5 to 8 cubic centi- 
meters) of filtered gastric juice in each of four test-tubes, add 
to each tube one or two pieces of the prepared albumin ; then. 

To tube 1 add nothing. 

To tube 2 add 1 drop of HCl. 



ESTIMATION OF PEPTIC ACTIVITY. 229 

To tube 3 add 4 grains of pepsin. 

To tube 4 add the above quantities of HCl and pepsin. 

These tubes are now placed in an incubator at 37° C, and 
from time to time examined to note the progress of liquefaction 
of the albumin. By comparison we can roughly determine 
whether digestion is progressing normally in the unaltered tube, 
and whether pepsin, h3^drochloric acid, or both are necessary to 
accomplish it. 

The results obtained from this investigation can only be 
considered in the light of an approximate indication of the peptic 
activity of the material tested. 

According to Nierenstein and Schiff,^ it is nesessary to dif- 
ferentiate three factors in order to arrive at an adequate idea 
of the significance of these pepsin determinations. These are: 
the diluting secretion, the pepsin secretion, and the hydrochloric 
acid secretion, all three of which must be regarded as distinct 
expressions of the secretory activity of the glandular parenchyma. 

Following a better knowledge of the activity of these dif- 
ferent factors, two improved modifications of the method of 
pepsin determination have been advanced. These are the meth- 
ods of Hammerschlag and of Mett.^ 

Method of Hammerschlag. — Briefly outlined, the method 
depends upon the activity of a few centimeters of gastric juice 
upon a 1 per cent, filtered solution of egg albumin. Two test- 
tubes, one filled with the albumin solution alone and the other 
with the albumin solution plus the gastric filtrate, are incu- 
bated for an hour at 37° C. At the expiration of this time the 
albumin content of each tube is estimated volumetrically, ac- 
cording to the method of Esbach (see page 324). The differ- 
ence between the albumin precipitate in the two tubes is equal 
to the amount of albumin digested, and, therefore, is a measure 
of the peptic activity of the gastric juice. The objections to this 
method are that the Esbach method is not very accurate, and 
also that albumoses are partly precipitated by the reagent. How- 
ever, this method is sufficiently accurate for the ordinarj^ clinical 
purposes, and when the Mett method cannot be followed may 
be adopted. It would be better, however, to employ a method 
which employs diluted gastric juice, as in the Mett method. 

5 Arch. f. Verdauungskrankh.. vol. iii, 1902. 

6 Sahli's : "Diagnosis," 1907. 



230 DETERMINATION OF FUNCTIONS OF STOMACH. 

Peepaeatiok of Mett^ Tubes. — Mix the whites of several 
fresh eggs and strain through cheesecloth^ select a number of 
10-inch lengths of thin walled 1 to 2 millimeters inside diameter 
glass tubings which should be thoroughly cleansed and dried. 
These are then sucked full of the albumin solution and kept in 
a horizontal position. Partly fill a large basin with water and 
bring to a boil, remove from the fire and then stir with a ther- 
mometer until the temperature falls to 85° C. Place the tubes 
immediately in this water, being sure to maintain them in a 
horizontal position^ and allow them to remain until the water is 
cool. The tubes thus prepared are filled with a hemogenous 
mass of soft-boiled egg-albumen. The tubes are then removed 
from the water, dried. and the ends sealed with sealing-wax or 
stick-lac, after which they will remain fit for use for a long 
time. 

These tubes are cut as needed into lengths of from 2 to 3 
centimeters. This cutting is best accomplished by nicking the 
glass with a small triangular file, when a quick bend at this 
point will usually cause accurate fracture of both tube and albu- 
min. Portions containing air bubbles or showing irregular 
fracture should be discarded. 

Teclmic."^ — For quantitative determinations of the peptic 
activity of the filtered gastric contents, employ a dilution of 
1 to 16, using as a diluent ^ HCl, using for each test 1 cubic 
centimeter of contents and 15 cubic centimeters twentieth normal 
HCl. 

This mixture is placed in a covered Stender dish^ after two 
Mett tubes have been placed in it, and the specimen allowed 
to digest in an incubator (37° C.) for twenty-four hours. At 
the end of this period the digested cylinders of Qgg albumin at 
each of the four ends are measured off and the average reading 
calculated. The relative amount of pepsin is then obtained by 
squaring the result (Shurtz's law), and if desired multiplying 
by the dilution, e.g., 16. For making the measurements a pair 
of calipers with a vernier reading 0.1 millimeter is con- 
venient. A lens is useful principally for reading the vernier. 



7 After Sailer and Farr, U. of Pa. Med. BuU., Oct., 1906. 

8 A Stender dish is similar to a Petri dish, but is deeper, with a flat ground- 
glass lid. 



ESTIMATION OF PEPTIC ACTIVITY. 231 

One end of the tube is placed against one jaw of the calipers, 
and the other is separated nntil its end is just visible through 
the opalescent edge of albumin. If the tube is at all oblique the 
shortest side is taken, while if the albumin is at all uneven, the 
highest point to which digestion has extended is taken. In place 
of the calipers the ordinary mechanical stage, which is usually 
fitted with a vernier scale, may be used. 

Nieren stein and Schiff have proven that a dilution of at 
least 1 to 16 is absolutely necessary if we wish to obtain a rela- 
tive idea of the quantity of pepsin as estimated from the diges- 
tion length. This is because with a dilution of less than 16, 
the length of digestion decreases as the length of time increases, 
owing to the concentration and activity of the inhibiting sub- 
stances. This great dilution also decreases the amount of pepsin 
in the mixture, so that the digestion length is kept within the 
limits of Shutz's law (3.6 millimeters in twenty-four hours). 
As there are instances when this dilution is not sufficient (with 
very active pepsin solution), it may become necessary, when 
the digestion length exceeds 3.6 millimeters with the 16-fold 
dilution, to repeat the pepsin test with a dilution of 1:32; then 
by squaring the length as before, and multiplying this quantity 
by 32, the relative amount of pepsin in the undiluted gastric 
contents is obtained. It is clear that in this estimation the unit 
of the relative amount of pepsin will be that quantity of pepsin 
by which 1 millimeter of albumin in the Mett tube will be 
digested in twenty-four hours with an acidity of 0.18 HCl. In 
this determination we do not consider the absolute quantity of 
pepsin, but simply the degree of concentration, for the result of 
Mett's method is the same whether large or small quantities of 
digestive substances are employed.^ 

Summary. — Conclusions of Sailer and Farr regarding varia- 
tions due to alterations in technic^^ : 

1. Difference in the caliber of the tubes. The variations in 
the reading of the two ends of one tube were as great as between 
tubes of different caliber for tubes between 1 and 2 millimeters. 
In tubes greater than 2 millimeters the rate of digestion seemed 
to be uniformly greater in tubes of larger diameter. 



9 Sahli's "Diagnosis," fourth edition, 1905. 
10 Sailer and Farr : hoc. cit. 



232 DETERMINATION OF FUNCTIONS OF STOMACH. 

2. The age of the tubes. Provided there is no separation 
of the albumin nor putrefactive softening, age does not seem to 
affect the tubes. The method of preparation tends to make them 
sterile. Tubes that are too fresh (less than four or five days 
old) are said to digest more rapidly than tubes that are "ripe." 

3. Variability in the digestion of albumin from different 
eggs is an unimportant factor. 

4. The degree of digestion within certain limits is said to 
vary directly with the duration of digestion. 

5. Variations in the temperature of the incubator. This is 
undoubtedly an important factor, but there are no definite data 
to offer. 

6. The effect of variations in the acidity of the original 
specimen. This has been provided against in the technic of the 
method, since the use of so large an amount of diluting acid 
solution tends to render the acidity of the diluted specimen 
uniform in every instance. 

DETERMINATION OF PANCREATIC ACTIVITY. 

The determination of pancreatic activity by the oil test- 
meal is based upon the experimental work of Boldiroff, who 
found that the introduction of oil into the stomach of dogs 
caused regurgitation of the duodenal contents in which pan- 
creatic ferments could be demonstrated. Volhard devised a 
method of demonstrating trypsin and applied it to clinical 
uses. 

This method has recently attracted sufficient attention to 
warrant a brief consideration of the method at this time, 
although the findings have not yet been proven conclusive. 

The clinical application as advocated by C. B. Farrii is as 
follows : — 

From 100 to 200 cubic centimeters of olive oil or cotton- 
seed oil are administered through the stomach-tube, into the 
fasting stomach. Food-remains or fluid if found in the stomach 
should first be removed through the tube. 

At the expiration of half an hour the contents of the 
stomach is aspirated; at this time there is usually recovered 



11 Jour. A. M. A., Dec. 11, 1909, 



DETERMINATION OF TRYPTIC ACTIVITY. 233 

only a few cubic centimeters of a whitish mucoid fluid ; in others 
as large an amount of pale green or dark green fluid is obtained. 

The tests are carried out upon the fluid without filtration. 

Eeliable material for the trypsin test is not always forth- 
coming, as^ while practically always some fluid is recovered, fre- 
quently this is nothing more than a small amount of retained 
or freshly secreted gastric juice or mucus. 

Farr suggests that the best criterion as to the character of 
the fluid should be its color, indicating the presence or absence 
of bile, and its response to the action toward ferments. I would 
suggest that the reaction would be also of great value in deter- 
mining the origin of the fluid. 

Methods. — Tlie Mett Method (fully described on page 
230) : This may be successfully employed, the only modifica- 
tion being the substitution of a decinormal solution of sodium 
carbonate for the HCl as described; particles of fibrin are placed 
in the dishes to detect tryptic digestion. 

Method of Gross. — A 0.1 per cent, solution of casein in a 
0.1 per cent, sodium carbonate solution is prepared and 10 cubic 
centimeters placed in each of several test-tubes. The trypsin 
solution serially diluted with water is added in 1 cubic centi- 
meter amount to the several tubes and all are incubated at 
37° C. for twenty-lour hours. At the end of this time 1 per 
cent, acetic acid is added to each of the tubes and the dilution 
noted in the tube in which cloudiness last appears. If the casein 
is completely digested no cloud appears. 

DETERMINATION OF TRYPTIC ACTIVITY. 

Spencer's Mdbthod:12 (a) Prepare five reagent tubes label- 
ing them from 1 to 5 ; to tubes 1 and 2 add 0.5 cubic centimeter 
of filtered gastric contents, (h) To tubes 2, 3, 4 and 5 add 0.5 
cubic centimeter of distilled water, (c) From tube 2 remove 
one-half of contents (0.5 cubic centimeter) .and place in tube 3. 
Eepeat this procedure bet^veen tubes 3 and 4 and 4 and 5, being 
careful to thoroughly mix the contents of each tube before the 
transfer of the 0.5 cubic centimeter. These dilutions are there- 
fore, 1, %, 1/4, % and YiQ. (d) To each tube add 1 drop of 
phenolphthalein indicator, then add drop by, drop a 2 per ceut. 

12 Spencer : Jour. Biol. Chem., 1915, xxi, 165. 



234 determi:n'ation of fuxctioxs of stomach. 

solution of soda bicarbonate until a pale but permanent pink 
color appears in each tube, (e) To tubes 1, 2. 3 and 4 add 0.5 
cubic centimeter of casein solution (0.4 gram casein in 40 cubic 
centimeters of X/10 XaOH) . to tube 5 add 1 cubic centimeter of 
casein solution. Xow add to each, tube 130 cubic centimeters of 
distilled water and 30 cubic centimeters of X/10 HCl. This 
leaves the solution alkaline to the extent of' 10 cubic centimeters 
of N/10 XaOH ; minus about 3 cubic centimeters neutralized by 
the casein. (/) Incubate for five hours at 40° C. (g) Pre- 
cipitate the undigested casein by drop by drop addition of the 
following solution : Glacial acetic acid 1 cubic centimeter, alco- 
hol (95 per cent.) 50 cubic centimeters, distilled water 50 cubic 
centimeters. The tubes in which digestion has been completed 
remain clear, the others become turbid. 

The determination of tr}'ptic power is expressed in terms 
of dilution. Thus complete digestion in tube 3 (dilution 14) 
shows four times the tryptic power of the undiluted gastric 
extract with a standard of 1, then the tryptic power would 
be 4. 

ESTIMATION OF THE ACTIVITY OF RENNIN OR 
MILK-CURDLING FERMENT. 

N'ormal gastric juice contains, besides hydrochloric acid 
and pepsin, the rennin ferment as a natural secretory product 
of the gastric mucosa. Eennin possesses the property of coagu- 
lating mUk without the presence or assistance of acids. 

Method of Leo. — To 10 cubic centimeters of fresh, 
uncooked neutral or amphoteric milk, add from 2 to 5 drops of 
filtered gastric jnice, and place the mixture in an incubator at 
37° C. If rennin is present in normal amount, curdling should 
take place m from ten to fifteen minutes. In this process the 
slight amount of acid contained in the gastric filtrate is insuffi- 
cient to cause coagulation. If curdling takes place very slowly 
it is questionable whether this change is due to the action of 
the rennin or to the formation of lactic acid, so to be exact the 
reaction of the mixture should be taken before and after curdling 
has occurred. The rennin reaction is certain to have occurred 
Only in the presence of an unchanged reaction. If coagulation 
does not occur within an hour rennin can be considered absent. 



GASTRIC CONTENTS. 235 

As a further guide it may be remembered that the characteristic 
curd from rennin is a cake of casein floating on clear serum, 
while the curd from lactic acid is lumpy and broken. 

DIGESTION OF STARCH AND SUGAR. 

During digestion starch is converted into grape-sugar, while 
cane-sugar is converted into invert-sugar (a mixture of cane- 
and grape- sugar) . 

Starch is recognized by the deep blue color produced by 
the addition of a dilute solution of iodine or LugoFs solution. 
This reaction grows less vivid as the starch is converted. If 
starch digestion is unduly delayed or does not occur, we may 
infer hyperacidity of the gastric juice. 

DETERMINATION OF THE RATE OF ABSORPTION 
FROM THE STOMACH. 

Penzoldt's Method. — A capsule containing potassium 
iodide (1% grains) is swallowed. The appearance of the iodine 
reaction in the saliva indicates that absorption has occurred 
from the stomach. To test for the iodide in the saliva, paper 
moistened with starch paste and dried is used. After the cap- 
sule has been swallowed, the paper is moistened with saliva at 
short regular intervals, and then touched with a glass rod dipped 
in commercial (better, fuming) nitric acid. Upon the appear- 
ance of iodine in the saliva the characteristic blue reaction 
occurs. 

When absorption is normal this reaction is usually positive 
in from ten to fifteen minutes ; but if absorption is delayed, the 
reaction may be slow in appearing or occur not at all. 

DETECTION OF BILE IN GASTRIC CONTENTS. 

Technic. — To 8 or 10 cubic centimeters of gastric filtrate 
add 5 to 10 grains of sodium sulphate (powdered) and thor- 
oughly saturate by shaking for at least two minutes. Add to 
this mixture 3 cubic centimeters of acetone and fully mix by 
inverting 5 to 10 times. Stand aside and allow the acetone to 
rise to the top in a distinct layer. This procedure extracts 
the bile pigment if present, which may then be demonstrated in 



236 DETERMINATION OF FUNCTIONS OF STOMACH. 

the acetone by the addition of a few drops of yellow nitric acid, 
which in the presence of bile will produce a green color in the 
acetone. 

A New Eeaction for Bile Pigments. — A. V. Torday and 
A. Klieris in working with the stomach-contents of a jaundiced 
patient found that the methyl-violet, with which they were 
testing for free hydrochloric acid, gave a red color instead of the 
usual blue. The urine of a jaundiced patient gave the same 
reaction. Investigating further, they found that various stain- 
ing fluids gave similar reactions with the bile-stained urine. 
Pure bile pigments have not been studied. They suggest the 
following : — 

Test. — The method consists of adding 1 drop of a 1 per 
cent, solution of the dye to 15 cubic centimeters of water, and 
to this 1 cubic centimeter of gastric filtrate. According to 
these observers, the delicacy of these tests was found to be about 
twice as great as the iodine or the Gmelin test. 

TEST OF THE MOTOR FUNCTION OF THE STOMACH. 

If attempted extraction of a full meal six hours after in- 
gestion fails when properly performed, or if nothing can be 
recovered from an Ewald test-breakfast after two and one-half 
hours, the motor function of the stomach may be considered 
normal. 

A SECOND METHOD is to administer a large dose (10 or 
15 grains) of salol and test the urine at definite periods for 
the appearance of the products of its decomposition. The com- 
ponents of salol, carbolic and salicylic acid, are separated in the 
alkaline juice of the small intestine. They remain unchanged 
and undissolved during gastric digestion. Salicylic acid is 
readily detected in the urine by the violet color produced by the 
addition of neutral ferric chlorid solution. This test is con- 
veniently performed by moistening filter-paper and bringing a 
drop of ferric chlorid solution in contact with it. If gas- 
tric peristalsis is normal, salicylic acid should begin to appear 
in the urine from forty to sixty or seventy-five minutes after 
ingestion of 15 grains of salol. 



13 Med. Record, Oct. 2, 1909. 



EXAMINATION OF STOMACH CONTENTS. 237 

Iodoform Method. — Give with the test-breakfast 1 gram 
of iodoform in a well-sealed capsule. The iodoform, being in- 
soluble in the gastric juice, will not be absorbed by the stomach, 
but will be passed on by peristaltic action to the intestine. By 
the action of the fluids of the duodenum the iodoform is decom- 
posed with the formation of soluble sodium iodid. The demon- 
stration of the iodid in the saliva by means of starch paper and 
nitrous acid will indicate the time when the gastric contents is 
being discharged into the intestine. lodin should normally be 
detected in the saliva in from one hour to one hour and a half 
after the ingestion of the capsule. 

BoAs's Method. — Boas administers a simple evening meal 
consisting of meat, bread and butter, and tea, washing out the 
stomach the following morning. If any food material is found 
the motor insufficiency is considerable. If the stomach be 
washed out previous to the administration of the evening meal, 
no food should be found in the stomach in the morning. 

INDIRECT EXAMINATION OF THE STOMACH 
CONTENTS. 

Gunzbueg's Method (see Hydrochloric Acid) . — A tablet of 
0.2 gram of potassium iodid is placed in a piece of the thinnest 
possible strongly vulcanized rubber tubing measuring about 2.5 
centimeters in length. The ends of the tubing are folded and 
the package tied with 3 threads of fibrin which have been 
hardened in alcohol. The package is now tested by placing it in 
warm water for several hours and examining the water for 
potassium iodid. The patient swallows one of these packages 
three-fourths of an hour after an Ewald meal, the saliva being 
tested for potassium iodid at intervals of fifteen minutes. In 
the presence of free hydrochloric acid, in normal amounts, the 
threads of fibrin are dissolved and the potassium iodid is ab 
sorbed, giving a reaction in the saliva in from one to one ana 
three-fourths hours. In cases of hypochlorhydria the reaction 
is delayed for six hours, indicating a practical absence of free 
hydrochloric acid. 

Sahli's Desmoid Eeaction. — Sahli has recently intro- 
duced the "desmoid bag'^ for use in estimating the functional 
activity of the stomach. These bags are made of the ordinary 



238 DETERMINATION OF FUNCTIONS OF STOIVIACH. 

rubber dam used by dentists and contain a pill of 0.05 gram of 
methylene-blue and 0.1 gram of iodoform. The bag is tied, in a 
manner specially outlined by Sahli, with catgut which has been 
allowed to dry, but which has been untreated chemically. This 
gut, according to Sahli, is digested only by the gastric juice and 
not by the pancreatic juice. This pill is administered to the 
patient immediately following the noon meal and the urine and 
saliva tested at intervals of one hour, beginning three hours after 
administration of the pill. The digestion of the gut by the gas- 
tric juice liberates the pill and permits of the absorption of both 
the methylene-blue and the iodoform. The methylene-blue will 
appear in the urine, coloring it green within six hours, while 
the iodine will be found in the saliva within two hours. Should 
the color of the urine not be distinctly green, this tint may be 
more clearly brought out by adding a few drops of acetic acid and 
boiling. Variations from the periods indicated above denote a 
hyperacidity or a hypoacidity of the gastric juice according as 
the time of appearance of the reactions is shortened or increased. 
As the gut is digested only by the gastric juice, a non-appearance 
of either reaction would indicate an anachlorhydria. 

RONTGEN RAY EXAMINATION. 

With the advent of the Eontgen ray and its practical appli- 
cation, it has become an invaluable aid to diagnosis. A number 
of competent men have applied this agent in the study of the 
digestive tract, and have reached so many valuable conclusions 
pertaining to the size, location, motility, etc., of the stomach 
that the examination of a patient suffering with any disorder 
of the digestive tract must be considered incomplete unless a 
rontgenologic examination has been made. The procedure and 
technic are omitted from this work, because the examination is 
beyond the field of the general practitioner, requiring, as it does, 
special technic, expensive apparatus, and a large experience in 
rontgenologic interpretations. 



IX. 

THE FECES. 



PHYSICAL CHARACTERISTICS. 

The Number. — One stool per day is the normal average for 
a healthy adult. Three daily, or one in forty-eight hours, may 
be normal for some individuals, and not incompatible with 
health. 

The Reaction. — Whether the stools be acid or alkaline is 
of no special clinical importance. In adults the reaction is usu- 
ally alkaline, sometimes neutral, but rarely acid. Acid stools 
are the rule in infants. 

The Amount. — The amount varies in proportion to the 
amount of solids ingested. A preponderance of vegetable food 
usually produces a large quantity, while animal food leaves com- 
paratively little residue. 

The average daily amount of feces varies between 60 and 
250 grams, of which 75 per cent, is water. 

To weigh solid feces ascertain the weight of both the feces 
and their container, then weigh the container empty, and sub- 
tract the latter from the former weight, which will represent 
the weight of the contained feces. If the feces are liquid they 
may be measured, and the amount expressed in cubic centi- 
meters. 

The Odor. — The disagreeable odor is largely due to the 
presence of indol and skatol, but may be further increased by 
hydrogen sulphide, methane, and methyl-mercaptan. 

The Consistence. — This varies greatly and depends largely 
upon the amount of fluids ingested, the temperature, the climate, 
and the condition of the digestive tract. In man the usual form 
is the characteristic plastic cylinder. Clinically expressed, the 
consistence of the feces may be liquid, mushy, or solid or formed. 

Sekous Stools. — These are composed of fluid without fecal 
matter, and are of considerable diagnostic importance. Such 

(239) 



240 THE FECES. 

stools are characteristic of Asiatic cholera, cholera morbus, chol- 
era infantum, and poisoning by antimony. In cancer of the 
rectum evacuations are small, frequent, and serous. Arsenic 
poisoning and acute catarrhal enteritis may produce this form 
of stool. Poisoning by toad-stools is also a -frequent cause of 
serous stools. 

The Color. — The color varies with the character of the food 
ingested, and is usually little influenced by the decomposition 
products of the biliary pigments. In adults, the color usually 
varies from a light to a dark-brown. Lack of color in the stools 
may occur in health, owing to the over-oxidation of the coloring 
matter into colorless products of bilirubin. Such stools do not 
necessarily indicate the presence of large amounts of fat nor 
obstruction of the biliary passages. 

In infants, fat and undigested milk produce a whitish-color 
tinged with bile-pigments. 

Green stools are seen after taking calomel. After expo- 
sure to the air such stools are usually acid. 

Yellow^ stools may be caused by the ingestion of santonin, 
rhubarb, or senna. T3^phoid stools are 3xllowish-brown and have 
received the name of "pea-soup stools.^' 

White or clay-colored stools frequently denote the ab- 
sence of bile, as in obstructive jaundice. 

A^ery dark or black stools may result from the administra- 
tion of iron, manganese or bismuth, or from the ingestion of 
much meat, blackberries or red-wine. 

"Tarry" stools usually denote hemorrhage. 

EXAMINATION OF INTESTINAL DIGESTION BY MEANS 
OF GLUTOID CAPSULES. 

Sahli recommends the use of glutoid capsules which are 
made from gelatin hardened in formaldehyde. These either do 
not dissolve in the gastric juice at all, or only after a consid- 
erable time, but are rather quickly soluble in the normal intes- 
tinal juices. 1 These are preferable to the keratin coated pills 
which were originally recommended, and are useful to diagnose 
the condition of intestinal digestion, e.g., the pancreatic func- 



1 Deutcti. mcd. V/och., No. 1, 1897. 



FUNCTIONS OF GASTRO-INTESTINAL TRACT. 241 

tion. Capsules are filled with some drug which does not diffuse 
through the capsule wall, and whose absorption may be studied 
by its appearance in the saliva and the urine. For diagnostic pur- 
poses glutoid capsules, containing 2 grains (0.13 gram) of 
iodoform and 4 grains (0.26 gram) salol^ are convenient. 

In order to make the conditions of the test as uniform as 
possible both as to the length of time the capsules remain in 
the stomach and the degree of digestive absorption, it is ad- 
visable to administer the capsule with the test-breakfast. 
Experience has shown that normally, under ideal conditions, 
i.e., normal gastric motility, normal intestinal digestion, and 
normal intestinal absorption, the iodin reaction may be expected 
to appear in the saliva, and salicyluric reaction in the urine in 
from four to six hours. 

If one wishes to test the accuracy of any particular lot of 
capsules, it will be necessary to prove that this reaction time is 
obtained in healthy normal individuals. Only rough approxi- 
mate differences in the time of the reaction are of clinical im- 
portance, so that it is sufficient to examine the saliva and the 
urine after six, ten, and twenty-four hours. The best results 
from this method are obtained by administering the capsule in 
the morning upon an empty stomach, and then, four hours later, 
allowing the patient to resume his meals as usual. The speci- 
mens of urine and saliva may, of course, be saved, and exam- 
ined afterward. 

DETERMINATION OF THE MOTOR FUNCTIONS OF 
THE GASTRO-INTESTINAL TRACT. 

Method of Adolph Schmidt. ^ — Schmidt demands two con- 
ditions for a satisfactory clinical method of examining the feces : 
1. A knowledge of what a normal stool should be under a certain 
diet, involving the use of a "test-diet." 2. The methods of 
examination must be so simple that they are within the reach 
of every physician. 

The Test-diet. — ^The requirements are: (a) That it be 
nutritious enough to furnish calories sufficient for the body's 
needs, (h) That it be so constituted that it can be readily ob- 



2 Quoted by Steele, Medical News, Dec, 1905. 

IG 



242 THE FECES. 

tained in any household or hospital dietary, (c) That it con- 
tains a known amount of its constituents, so that variations in 
digestion and absorption can be detected in the stool. 

Schmidt suggests the diet which is given below, followed 
by the approximate equivalents as suggested by Steele : — 

Milk, 1.5 liters {2% pints). 

Zwieback, 100 grams (3 ounces) well-dried toast. 

Two eggs. 

Butter, 50 grams (1% ounces). 

Beef, very rare or raw, 125 grams (3^ pound). 

Potatoes, 190 grams (6 ounces). 

Gruel made from 60 grams oatmeal (2i/2 ounces). 

Sugar, 20 grams (% ounce). 

This may be given somewhat as follows : — 

Breakfast. — ^Two eggs, half of the amount of toast and 
butter, 2 glasses of milk, oatmeal, and sugar. 

Dinner. — The steak and potatoes, % of the amount of toast 
and butter, 1% glasses of milk. 

Supper. — ^Two glasses of milk, remainder of toast and 
butter. 

This diet is sufficient for the needs of the average patient, 
and, while not offering much variety, is only taken for two or 
three days. A capsule containing 5 grains of charcoal is given 
with the first meal, and no examination made until the charcoal 
(black) appears in the stool. 

The amount of each article composing the day^s dietary 
must first be accurately determined and compared to the con- 
tents of convenient measures, so that the articles need not be 
weighed every meal. This does not apply to the meat and 
potatoes, which should be weighed each day. 

The Period of Passage. — The period of time required for 
food to pass through the intestinal tract can easily be deter- 
mined by watching for the black in the stool. It is quite as 
necessary to know the time required for the passage of chyme 
through the gastro-intestinal tract as it is to ascertain how 
often the stools occur, since the two are in no way identical. 

It has been shown in chronic colitis with several watery 
stools a day that the period of passage may be normal, and that 
peristalsis is decidedly increased only in the colon. According 



FUNCTIONS OF GASTROINTESTINAL TRACT. 243 

to Strass, using a diet similar to Schmidt, the time required for 
food to pass through the alimentary canal varies normally be- 
tween ten and thirty hours. Under abnormal conditions this 
may vary between four and forty-eight hours. 

The Specimen. — After the appearance of the charcoal, 
which indicates the beginning of the passage of the test-diet, 
a portion of one passage is collected in a clean receptacle and 
transferred to a clean, wide-mouthed bottle, and conveyed to the 
laboratory for examination. 

Much can be determined from a naked-eye examination of 
such a mass. The technic for this examination, as outlined by 
Steele, is as follows : — 

Take a piece of formed stool about the size of an English 
walnut or an approximate equivalent of liquid feces, and rub it 
up in a mortar with distilled water until quite smooth and 
liquid. Part of this is then poured into a Petri dish and 
examined in a good light on a dark background. A little 
experience will enable the observer to acquire a large amount 
of information about the composition of the stool by this 
macroscopic examination. 

The Gross Appearance of Normal Stools. — In normal diges- 
tion nothing should be seen by the naked eye, save a varying 
number of brown points (oatmeal husks), cellulose and indiges- 
tible parts of food, and occasionally sago-like grains that look 
like mucus, but which are found upon microscopic examination 
to be grains of potato. If, on the addition of the water, fat 
globules are seen, they must be considered pathologic unless the 
patient is known to have ingested excessive amounts of fats, as 
olive or castor oil. 

Gross Appearance of Patholo^c Stools.— (a) Mucus in 
large or small flakes is not affected by rubbing in the mortar. 
The mucus appears as glassy, translucent flakes often stained 
yellow by bile-pigment. Doubtful cases may be decided by the 
microscope. 

(b) Pus in sufficient quantity to be recognized macro- 
scopically will have the characteristics of pus in any other 
region. It usually appears as small, yellowish streaks or collec- 
tions throughout the stool. 



244 THE FECES. 

(c) Blood in appreciable amount, if from high in the di- 
gestive tube, will color the stool dark reddish brown or black; 
becoming more nearly the color of fresh blood as the source of 
origin approaches the lower end of the bowel. 

(d) Parasites. — Segments of tape-worm, also thread- and 
seat-worms, may be detected in varying numbers in patients so 
afflicted. 

(e) Foreign bodies having entered the digestive tract, if 
indigestible and not impeded in their progress, may be recovered 
from the stool at varying periods after ingestion. 

(f) Calcidi from the various glands situated along the di- 
gestive tract, or from the intestinal canal itself (enteroliths), 
may be found in the feces, and appear as calcareous masses of 
various sizes and composition, depending upon their origin and 
age. 

(g) Bemnants of connective tissue and sinew from beef- 
steak. These can be detected by their whitish-yellow color and 
toughness, by which they can be distinguished from mucus. In 
case of doubt the piece should be examined microscopically with 
a drop of acetic acid. Connective tissue then loses its fibrous 
structure, while mucus becomes more thread-like. Small, single 
pieces of connective tissue can be found in normal stools, 

(h) Bemnants of Muscle-fiber. — These appear as small 
reddish-brown threads or small, irregular lumps. 

(i) Bemnants of Potato. — These appear like boiled grains 
of tapioca and may easily be confused with mucus. The micro- 
scope will show the true nature of these bodies. 

(j) Large crystals of triple phosphate occur in foul stools, 
and can be recognized by their shape and by their solubility in 
all acids. 

Microscopic Appearance of Normal Stools. ^ — Three slides 
are prepared from the liquid feces. The first is merely a drop 
of the material to be examined by both low and high power. 
The second slide is prepared by mixing a drop of acetic acid and 
a drop of the liquid feces upon a slide, heating it to boiling and 
then putting on a cover-glass. The third slide is prepared by 
mixing a drop of liquid feces with a drop of Lugol's solution. 



3 Technic of E. Button Steele, Medical News, Dec. 16, 1905. 



CHEMICAL EXAMINATION. 245 

Microscopic Examination of Slides. — Slide 1, — will 
show (a) single small muscle fibers colored yellow with cross 
striations. Visible with a Leitz 3, but showing better with higher 
power, (h) Small and large yellow crystals of salts of the fatty 
acids, (c) Colorless (gray) particles of soap, (d) Single potato 
cells without distinguishable contents, (e) Particles of oat- 
meal and grain husks. 

Slide 2. — A general idea of the fat-content of the stool may 
be obtained. Upon cooling, small drops of fatty acids may be 
found covering the whole preparation. The large crystals of 
salts of the fatty acids are broken up by the acetic acid, and 
fatty acids liberated. If the slide is heated again and exam- 
ined hot, the fatty acids will be found to run together in drops, 
which, as the slide cools, break suddenly apart. 

Slide 3. — Here should be found violet-blue grains in some 
of the potato cells, and small single blue points, probably fungi 
or spores. 

Pathologic Microscopic Findings. — Slide 1. (a) Muscle 
fibers in excess, perhaps with yellow nuclei, (h) Neutral fat 
drops, or fatty acid, in soap crystals, (c) An excess of potato 
cells with more or less well preserved contents, (d) Parasite 
eggs, mucus, connective tissue, pus, etc. 

Slide 2. — Fatt3^-acid droplets in excess. 

Slide 3. — Blue starch grains in the potato cells or free oat- 
meal cells, fungi, spores or mycelia. 

Formed Elements in the Feces. — (a) Blood. Eed cells, 
if recently shed, may be distinguished by their characteristic 
form, or if disintegrated, only masses of brownish-red amorphous 
hematoidin will be found. In a certain percentage of cases char- 
acteristic crystals of hematoidin will be found. 

(b) Epithelial Cells. — These are normally present in mod- 
erate numbers and represent the natural desquamation from the 
intestinal canal. They are more or less disintegrated, depending 
to some extent upon their height of origin in the intestinal canal, 
and upon the length of time they have remained free in the 
digestive tube. In catarrhal conditions they may be present in 
very large nnmbers, when they may assume diagnostic impor- 
tance. 



246 THE FECES. 

(c) Pus Cells. — These rapidly undergo decomposition, so 
that even when numerous and coming from a comparatively 
short distance above the rectum, they may be beyond recogni- 
tion. The characteristic pus cell appears as a small round or 
slightly oval granular body. The presence of very much pus in 
the stools is indicative of rupture of an abscess into the intes- 
tinal tract. 

METHOD FOR MICROSCOPIC EXAMINATION OF 
EXTRACTS OF FECES. 

Smithies^ describes a procedure, recently adopted at the 
Mayo clinic, intended to increase the value of the routine micro- 
scopic examination of feces. A 2 per cent, agar jelly is first 
made by boiling strip agar in distilled water and filtering several 
times while very hot. The product is sterilized and kept for con- 
venience in test-tubes each containing 5 cubic centimeters. In 
examining a specimen of feces emulsion, the agar jelly is lique- 
fied by heating, 2 cubic centimeters of it are poured into each of 
two small test-tubes, and 15 drops of filtered staining agent then 
added. For staining bacteria, epithelia, etc., Unna's polychrome 
methylene-blue is used ; for starch elements and vegetable fibers, 
LugoPs solution. Thin smears of the feces are made on cover- 
slips, dried, covered with 1 drop of the agar stain mixture, and 
mounted on slides. As the agar cools it solidifies, while the stain 
mixed with it permeates the smear. The agar gives so firm a 
mount that the specimens may be examined with high power and 
the stage of the microscope at any angle. The mount is suffi- 
ciently permanent to allow of future study of the specimens. By 
using the agar without any stain, motile micro-organisms may be 
observed for a long time. 

Schmidt's Test for Pancreatic Insufficiency. — This test is 
based upon Schmidt^s contention that the nuclei of cells are 
digested only by the pancreatic enzyme and therefore the appear- 
ance of nuclei in the feces indicates reduced or absent pancreatic 
activity. 

Techxic. — Prepare the test-material by cutting fresh beef 
in cubes about i/o centimeter square, harden in alcohol and tie 
np in small pieces of fine gauze. These may be kept in alcohol 



4 Archives of Internal Medicine, June, 1912. 



EXAMINATION OF EXTKACTS OF FECES. 247 

until used. Before using the bags should be well washed in 
water for several hours. 

Limit the test-diet by the administration of charcoal or lyco- 
podium (5 to 15 grams in capsules), then have the patient sav al- 
low several of the prepared bags with the test-diet during twenty- 
four hours. After the stool marker appears, sieve a portion of 
stool to recover the test bags, open and examine the contents for 
nruclei, either fresh or after treating with acetic acid or methy- 
lene blue solution. In doubtful cases Schmidt recommends pre- 
paratory hardening and staining of the recovered material. 

Method of Gross For Tryptic Activity. — ^Eub up a small 
portion of feces in a mortar with 3 parts of solution b, (for 
reagents see Appendix, page 497), filter and refilter until the 
filtrate is clear, place 10 cubic centimeters of the fecal filtrate 
in a small flask with 100.0 cubic centimeters of solution a to 
which a few cubic centimeters of toluol are added to prevent 
bacterial growth. The flask is then placed on an incubator at 
37° C. From time to time small portions are removed and 
tested for the presence of casein as follows : — 

Casein is precipitated by 1 per cent, solution of acetic acid 
while the products of its digestion are not. Proceed to inter- 
mittently test a small portion removed from the flask from time 
to time until the casein has disappeared from the solution. Note 
the time of absence of precipitation from the beginning incuba- 
tion. The rapidity of digestion varies with the diet. It is most 
rapid after a proteid diet, therefore, the test for the presence of 
trypsin should be made after such a diet. The normal limits of 
complete digestion of this test are eight to fifteen hours. 

"Wohlgemuth' s Method of Determining Amylolytic Activity 
as Modified by Hawk.^ — ^Weigh accurately about 2 grams of 
fresh feces into a mortar (duplicate determinations should be 
made), and add 8.0 cubic centimeters of phosphate-chlorid solu- 
tion [0.1 mol. dih3^drogen sodium phosphate, and 0.3 mol. di- 
sodium hydrogen phosphate per liter of 1 per cent, sodium 
chlorid (see page 501)], 2 cubic centimeters at a time, rubbing 
the feces mixture to a homogeneous consistency after each addi- 
tion of the phosphate-chlorid solution, allow the mixture to 

5 Hawk : Arch. Int. Med., 1911, viii, 552. 



248 THE FECES. 

stand at room temperature for half an hour with frequent stir- 
ring. This produces a neutral fecal suspension. Transfer this 
to a graduated 15.0 cubic centimeter centrifuge tube, washing 
the mortar and pestle carefully with the phosphate-chlorid solu- 
tion and add the washings to the contents of the centrifuge tube, 
finally filling up to the 15 cubic centimeter mark with the same 
solution. Eotate for fifteen minutes or more to secure satisfac- 
tory sedimentation. At this point read and record the height of 
the sediment column. Eemove the supernatant liquid by means 
of a bent pipette, transfer it to a 50-cubic centimeter volumetric 
flask and dilute to the 50 cubic centimeter mark with the phos- 
phate-chlorid solution. Mix the fecal extract thoroughly and 
determine its amylolytic activity. 

Technic. — For this purpose a series of six graduated tubes 
are prepared, containing volumes of the extract ranging from 2.5 
to 0.078 cubic centimeters, each of the intermediate tubes in 
this series will thus contain one-half as much fluid as the pre- 
ceding tube. ]N"ow make the contents of each tube 2.5 cubic 
centimeters by means of the phosphate-chlorid solution. This 
secures a uniform electrotyte concentration. Introduce 5.0 
cubic centimeters of a 1 per cent, soluble-starch solution and 3 
drops of tolulol into each tube, thoroughly mix the contents by 
shaking, close the tubes by means of stoppers and place all in 
incubator at 37° C. for twenty-four hours. 

To prepare the 1 per cent, starch solution, the weighed 
starch powder should be dissolved in cold water in a casserole 
and stirred until a homogeneous suspension is obtained. The 
mixture should then be heated with constant stirring until it is 
clear. This ordinarily takes from eight to ten minutes. A 
slightly opaque solution is thus obtained, which should be cooled 
and made up to the proper volume by adding water (before 
using). At the end of twenty-four hours remove the tubes, fill 
each to within % inch of the top with ice water, and 1 drop 
of N/10 iodine solution, thoroughly mix the contents and 
examine the tubes carefully with the aid of a strong light. 
Select the last tube in the series which shows entire absence of 
blue color, thus indicating that the starch has been completely 
transformed into dextrin and sugar, and calculate the amylase 
activity on the basis of this dilution. In case of indecision be- 



EXA]\IINATION OF EXTRACTS OF FECES. 249 

tween two tubes, add 'an extra drop of the iodine solution and 
observe them again. 

The am3dolytic activity, Df, of a given stool may be ex- 
pressed in terms of 1 cubic centimeter of sediment obtained by 
centrifugation, as above described. For example, if it is found 
that 0.31 cubic centimeter of the phosphate chlorid extract of 
the stool acting at 38° C. for twenty-four hours completely 
transformed the starch in 5 cubic centimeters of a 1 per cent, 
starch solution then we would have the following proportion : — 
0.31 : 5 : : 1 (c.c. extract) : x. 

The value of x in this case is 16.1 which means that 1 cubic 
centimeter of the fecal extract possesses the power of completely 
digesting 16.1 cubic centimeters of 1 per cent, starch solution 
in twenty-four hours at 38° C. 

Inasmuch as stools vary so much as to water content, it is 
essential to an accurate comparison of stools that each compari- 
son is made on the basis of the solid matter, supposing for 
example, that in the above determination we had 6.2 cubic 
centimeters of a sediment, since the supernatant fluid was re- 
moved and made up to 50 cubic centimeters before testing its 
amylolytic value, it is evident that 1 cubic centimeter of this 
sediment is equivalent to 8.1 cubic centimeters of extract. There- 
fore, in order to determine the amylolytic value of 1 cubic centi- 
meter of sediment, we must multiply the value (16.1) as ob- 
tained above for the extract by 8.1. This yields 130.4 and en- 
ables us to express the activity as follows : — 

38° 

Df =130.4 

24 

Wohlgemuth finds that the normal average value is 
about 150. 

CHEMICAL EXAMINATION. 

This comprises only five routine tests : 1. The reaction. 
2. The sublimate test for the condition of the bile salts. 3. The 
fermentation test. 4. The test for "lost albumin." 5. The test 
for occult blood. 

1. The Reaction. — This is quite difficult to get with the 
ordinary litmus paper. It can be easily determined by dropping 



250 THE FECES. 

a littk softened fecal matter into 5 or 10 cubic centimeters of 
weak watery solution of neutral litmus, shaking it and noticing 
the color reaction. 

2. The Sublimate Test. — This consists of taking a few 
cubic centimeters of liquid feces and mixing with an equal 
amount of a saturated watery solution of HgCl2. A normal 
stool will quickly turn a pinkish red, indicating the presence of 

■ hydrobilirubin, which will be more intense the fresher the 
material. A green color is pathologic and indicates the presence 
of unchanged bile-pigment. 

3. Fermentation Test. — Described by Steele, using his 
modification of Strasburger's apparatus. 

The apparatus consists of a 2-ounce, wide-mouth bottle 
A (see Fig. 56). This is fitted with a perforated cork through 
which runs a tube to the test-tube B, which is also fitted with 
a rubber cork with two perforations. A bent tube runs from 
the tube B to the test-tube C, to allow for the escape of air. 
Each tube has a capacity of a little more than 30 cubic centi- 
meters when fitted on the corks. The apparatus is simple and 
easily constructed, and broken parts can be replaced readily. 

The Test. — About 5 grams of solid feces, or an equivalent 
of liquid feces, are rubbed up with a little distilled water and 
placed in the main bottle A. This is filled with sterile water, 
the tube is filled with water and fitted into place (not neces- 
sarily full), and tube C is then fitted on empty. The reaction 
IS carefully noted before the test is started. The apparatus is 
then stood in a warm place — ^best in an incubator at 37° C. for 
twenty-four hours. If gas forms by fermentation in A it will 
rise into B, and the amount will be indicated by the amount of 
water displaced into C. Normally the fermentation test should 
show practically no gas, and the original reaction of the material 
should be unaltered in twenty-four hours. If more than ore- 
third of the tube C is filled, it is pathologic. If the reaction 
after twenty-four hours is decidedly more acid, it is a carbo- 
hydrate fermentation. If alkaline, and with a foul smell, it is a 
fermentation of the albumins. 

4. Test for Lost Albumin. — A portion of softened stool 
is filtered (a slow and difficult process). The filtrate is shaken 
with silicon and refiltered, then is saturated with acetic acid to 



CHEMICAL EXAMINATION. 



251 







^^^^^^ 



Fig. 50.— Strasburoek Aitauatus, Showinpx Aurangkmext of Bottles 
FOR Fermentation Test of Feces. (After Steele.) 



252 THE FECES. 

bring down the niicleo-proteids, and finally a drop of potassium 
ferrocyanide solution is added. A decided precipitate indicates 
albumin. A positive test shows only that there is a decided 
diminution in albumin digestion. 

Total Fats of Undried Feces. — Method of Saxon^ : This is 
essentially a combination of the method of Folin and Went- 
worth" for the determination of the total fats of powdered dried 
feces, and the method of Meigs^ for the determination of fat of 
milk. 

Techxic. — Collect a twenty-four hour specimen of feces 
and very thoroughly mix until a homogeneous paste results. If 
the stool is liquid, infusorial earth is slowly added, after weigh- 
ing the specimen, while mixing, until a workable paste is 
formed. The mass is weighed before and after a sample for ex- 
traction is chosen. I:^ the mixing has been done in an ordinary 
mortar it is advisable to remove the mass quantitatively to 
waxed paper for weighing. 

The sample for extraction is placed in a 100 cubic centim- 
eter glass-stoppered, graduated cylinder. Care must be taken 
not to smear the neck of the cylinder. This may be avoided by 
removing small portions from various parts of the mass with the 
aid of short capillary tubes sealed at both ends. The tubes and 
the portions of specimen may be as much as 5 or 6 gTams. 

Add 20 cubic centimeters of distilled water and 1 to 2.5 
cubic centimeters of concentrated HCl (depending on the 
amount of the sample) and again sufficient water to make a 
total bulk of 30 cubic centimeters. Add exactly 20 cubic 
centimeters of ether and shake vigorously for five minutes. 
Allow to stand a few seconds, remove stopper and add exactly 
20 cubic centimeters of 95 per cent, alcohol and again shake 
vigorously for five minutes. 

Stand the cylinder aside. The ether will extract practically 
all the fat which will come toi the top as a colored transparent 
layer. The ether layer is blown off into a tall 150- or 200- cubic 
centimeter beaker. This is accomplished in the same manner 



6 Saxon : Jour, of Biol. Chem., xvii, 2, 1914. 

" Folin and Wentwortli : Jour, of Biol. Chem., vii. p. 421, 1910. 

s Arthur V. Meigs : Phila. Med. Times, July 1, 1S82. Arthur V. Meigs 
and Howard L. Marsh : The Medical Record, Dec. 30, 1911. Croll : Biol. 
Chem. Bull., June, 1913. 



CHEMICAL EXAMINATION. 253 

that water is blown from a wash-bottle. The submerged end of 
the delivery tube- is bent upward as in the pipette described by 
Meigs and Marsh^ in order that there be no disturbing currents 
which would set in motion the subjacent alcohol-water- feces 
layers. In this manner practically all the ether can be removed. 
The thin layer which remains is diluted with 5 cubic centim- 
eters of ether^ slightly agitated and blown off. This is done 
in all five times, care being taken each time to wash down the 
sides of the cylinder. The stopper also should be washed. 

Twenty cubic centimeters of ether are again added, and 
the cylinder shaken for five minutes and set aside. When the 
ether has stratified, blow it off and wash as before. During the 
second washing process the stratification should be allowed to 
complete itself. Evaporation is carried on until no trace of 
alcohol which ha^ been carried over by the ether remains. 
Ftom this point on the method is that of Folin and Wentworth. 
To the residue add 30 cubic centimeters of low-boiling petroleum 
ether (should distill over below 60° C.) and allow to stand over 
night. Petroleum ether for this work should be frequently 
tested for residue on evaporation. If a residue is left the ether 
should be redistilled. 

Filter the fatty petroleum ether, catch filtrate and wash- 
ings in a tall, weighed, 100-cubic centimeter beaker, evaporate 
off the solvent, dry the beaker at 90° C, desiccate and weigh. 
Subtract the weight of the beaker from the last weighing and 
the result is the weight of neutral fat, free fatty acids and the 
fatty acids of the soaps contained in the specimen extracted. 

The fatty acid titer is obtained by dissolving the contents 
of the beaker, after the weighing, in 50 cubic centimeters of ben- 
zol, heating almost to the boiling point, adding 2 drops of a 
0.5 per cent, alcoholic solution of phenolphthalein and titrating 
with a decinormal solution of sodium alcoholate. Each cubic 
centimeter of the standard solution used in the titration repre- 
sents 28.4 milligrams of stearic acid. 

The difference between the gTavimetric and the volumetric 
determinations is the weight of tlie neutral fat. In the prepara- 
tion of the sodium alcoholate solution, absolute alcohol and 



Meigs and Marsh : Jour, of Biol. Chem., xvi, 152, 1913. 



254 THE FECES. 

freshly cut bright metallic sodium are used; otherwise it is the 
same as the standardization and preparation of any other 
standard solution. 

BLOOD IN THE STOOL. 

If bleeding has its origin in the upper part of the digestive 
tract (stomach or small intestine), it is so altered by the action 
of the digestive fluid that, by the time it finally appears in 
the stool, it has a black or brownish-black appearance which has 
been likened to tar or coflee grounds. However, if the hemor- 
rhage be large and the peristalsis very active, blood may appear 
almost unchanged in the stool, even when of high origin in the 
digestive tube. 

Eelatively small amounts of blood may be so changed and 
mixed with the feces that they cannot be detected by the naked 
eye or by the microscope. This is termed ^^occult" blood, and 
is only recognizable by chemical means. 

Preliminary Technic. — Owing to the possibility of the pres- 
ence of a positive reaction, resulting from the ingestion of hemo- 
globin-containing food in a normal individual, it becomes neces- 
sary to restrict the diet in order to eliminate this possible source 
of error, and to limit the test-diet by the administration of a 
capsule containing 5 or 10 grains of charcoal, and to watch 
the stools for the appearance of the black discoloration due to 
the passage of the charcoal. Only after the appearance of the 
charcoal in the feces should a specimen be taken for the test for 
occult blood. 

Steele^o recommends a "liquid diet,^^ including milk and 
broths, or a "semi-liquid diet^^ composed of milk, eggs, and toast, 
to which may be added moderate amounts of the ordinary winter 
vegetables. 

Eed meats and beef juice should positively be withheld. 
Steele has shown that iron, either in the organic or inorganic 
form, will even in large doses not aflect the reaction. 

As a preliminary to the test, it is necessary to eliminate as 
fully as possible extraneous sources of blood. Thus, tuber- 
culous ulcer, typhoid fever, hemorrhoids, fissure, and pur- 

lOAmer. Jour. Med. Sci., July, 1905. 



BLOOD IN THE STOOL. 255 

pura should be excluded. Also ingestion of carmine, swallowed 
blood from any cause, hemoptysis, epistaxis, and menstruation. 
Qualitative Detennination of Blood in Feces. 

GuAiAC Test. 11 — If the feces are solid they must be 
softened with distilled water. To 5 or 6 cubic centimeters of 
liquid feces, in a wide-mouthed cork-stoppered bottle, add about 
thrice as much ether and agitate or thoroughly mix by shaking. 
Then add a few grains of powdered guaiac and again agitate. 
Follow this by 5 cubic centimeters of glacial acetic acid (99.4 
per cent.), and still again agitate. Allow this mixture to stand 
until the solid particles settle to the bottom, and then decant into 
each of two test-tubes 5 cubic centimeters of the supernatant 
liquid. 

One test-tube should be kept as a control. To the other add 
1 or 2 cubic centimeters of a fresh solution of hydrogen dioxid. 
If a bluish discoloration occurs either at the line of contact of 
the two solutions or throughout the mixture, the reaction is 
positive. 

Van Deen's Guaiac Test. — ^Prepare 5 to 10 cubic centi- 
meters of an aqueous suspension of feces; add one-third of its 
volume of glacial acetic acid. Shake well in a large test-tube 
and add 3 to 5 cubic centimeters of ether. Again shake and 
allow to stand. The ether will rise to the top and in the pres- 
ence of blood will assume a brownish color. If the ether extract 
is turbid it may be cleared by the addition of a few drops of 
alcohol. Dissolve a few grains of powdered guaiac in 95 per 
cent, alcohol. To 5 cubic centimeters of this add an equal 
amount of ozonized turpentine and then overlay with the 
ethereal extract of feces. In the presence of blood a blue ring 
will form at the line of contact. Much blood will impart a 
blue color to the whole mixture when shaken. 

Precautions to be observed in chemical examinations for 
blood : — • 

Avoid using tubes containing traces of copper or nitric 
acid as traces of these may give a positive reaction (Webster). 

Remember, certain vegetables as watermelon and prunes, 
also medicinal iron and much pus may give a distinct positive 
reaction. 



11 Technic employed in Dr. J. Daland's Laboratory. 



256 THE FECES. 

According to Webster boiling the watery extract before pro- 
ceeding with the test will obliterate these sources of error. 

Klunge's Aloin Test. — According to Boas, the guaiac test 
for blood does not always yield decisive results in the examina- 
tion of the feces, since the blue color is often veiled by brown 
shades and rendered indistinct. For this reason a control test 
with aloin, as suggested by Klunge and others, is desirable. This 
test is performed as follows: 5 cubic centimeters of the stool 
are extracted with 20 cubic centimeters of ether, in order to 
remove the fat, which would subsequently interfere with the test 
by the formation of emulsions. After the removal of the ether 
3 to 5 cubic centimeters of acetic acid are added to the feces, and 
the mixture is again extracted with ether in a test-tube. The 
acetic acid-ethereal extract obtained in this manner is then em- 
ployed for the investigation. A solution of aloin is prepared by 
dissolving as much aloin as can be placed upon the point of a 
small knife in from 3 to 5 cubic centimeters of 60 to 70 per cent, 
alcohol. To the acetic acid-ethereal extract is first added 20 to 
30 drops of a resinous oil of turpentine, and then 10 to 15 drops 
of the solution of aloin. If the stool contains blood the resulting 
mixture soon becomes bright red, and upon standing for a time 
assumes a cherry color. If no blood be present the aloin solution 
remains yellow for at least one or two hours, and then acquires a 
slightly reddish tinge. According to Boas, the aloin reaction may 
be markedly accelerated by the addition of a few drops of chloro- 
form. With this modification, agitation of the mixture results 
in the formation of reddish droplets, which settle in the bottom 
of the tube as an intense red precipitate. According to Brand- 
berg, the oil of turpentine may be replaced by a dilute solution of 
hydrogen peroxid. 

Benzidin Test. — A few granules of benzidin are dissolved 
in 2 cubic centimeters of glacial acetic acid. A small fragment 
of the stool is mixed with 2 cubic centimeters of water and 
boiled in a test-tube. Ten drops of the benzidin-acetic acid 
solution and 3 cubic centimeters of 3 per cent, hydrogen 
peroxid are mixed in a test-tube and a few drops of the cooked 
emulsion of feces then added, A greenish or bluish color 
shows the presence of blood. The ethereal extract of the pre- 



BLOOD IN THE STOOL. 257 

vious test may also be used, adding the benzidin-acetic acid and 
peroxid. 

This reaction is extremely sensitive and care should be 
taken to see that the patient is not eating meat. The benzidin 
may be dried on a paper, as suggested by Einhom, and the 
boiled stool mixed with a little peroxid and dropped on the paper. 
The blue color should appear within two minutes. 

R. F. Ruttan and R. H. M. Hardistyi^ say that in orthoto- 
lidin we have a reagent for occult blood that is more satisfactory 
than benzidin on account of its greater delicacy, its more lasting 
color, and the fact that it can be made into a solution that re- 
tains its delicacy unimpaired for three or four weeks. The sub- 
stance known as tolidin, or orthotolidin, is a crystalline, basic 
body of the aromatic series with melting point from 130° to 130° 
C, very soluble in water, easily soluble in alcohol and ether, and 
closely allied to toluidin and benzidin. They assert that it is 
superior to the reagents in general use for clinical work. While 
benzidin is equally good for the detection of blood in the feces 
and stomach contents, this is true only for freshly prepared solu- 
tions of benzidin, solutions of which lose 50 per cent, of their 
delicacy in twenty-four hours, while tolidin remains unchanged 
for three or four weeks. Another point in favor of tolidin is 
that when the blood is in small quantity the reaction increases 
gradually in intensity and persists longer than with the other 
reagents. 

Modified GtUAiac Test. — L. de Jager,!^ {^^ order to bring 
out more plainly the blue color denoting a positive result in the 
guaiac test for blood in the feces proceeds as follows : A piece 
of the feces the size of a pea is inserted in a large test-tube with 
30 per cent, acetic acid and the tube filled about two-thirds with 
the acid. Ether is then added, the tube shaken up and allowed 
to stand until the ether rises to the surfaces; the addition of a 
few drops of alcohol, together with gentle stirring, will hasten the 
separation of the ether. To 5 cubic centimeters of the ether 
solution are then added 5 drops of a 20 per cent, sodium hy- 
droxid solution, 10 drops of freshly prepared guaiac solution, and 
2 cubic centimeters of a 3 per cent, solution of hydrogen per- 

12 Canadian Medical Association Journal, November, 1912, 

13 Zeutralblatt fiir innere Med., June 22, 1912. 

17 



258 THE FECES. 

oxid. Where a small amount of blood is present there develops 
a distinct green color ; if the amount is large, indigo blue. Thus 
performed, the guaiac test was found to be more sensitive than 
where the hydroxid had not been added, 4 parts of blood in 
100,000 giving a distinctly positive and prompt result, as against 
6 to 100,000 where the alkali is omitted. 

PHENOiiPHTHALMisr Test. — This test, originally employed 
by Meyer and later by Utz, has been so improved by Kastle, 
Amoss, and Benoit that it is now probably the most delicate 
one for the detection of blood, especially in stains, now avail- 
able, its limit of delicacy being about 1 part of blood in 8 
million of water. As in the other tests, the hemoglobin acts as 
an oxygen carrier, the active oxidizing agents being ozonized 
turpentine or hydrogen peroxid. 

Phenolphthalin is a product of the reduction of phenol- 
phthalein by zinc in alkaline solution. When oxidized in alkaline 
solution, it is converted into phenolphthalein with production of 
an intense red color. The reagent may be obtained in the market 
or, preferably, prepared as follows: Phenolphthalein is dis- 
solved to considerable excess of 30 per cent, sodium hydrate 
solution and boiled with an excess of zinc dust until a few 
drops of the strongly alkaline liquid no longer gives a red color 
after neutralization with HCl and sufficient alkali to alkalinize 
the solution. The solution is then decanted from the excess 
of zinc dust and the phenolphthalin is precipitated by acidify- 
ing with HCl. Collect the precipitate on a filter and purify 
by repeated crystallization from water and alcohol. This puri- 
fication is continued until a white, crystalline compound is 
obtained free from every trace of phenolphthalein (as shown 
by absence of red coloration on addition of alkali). Dry at 
room temperature or in the oven at 50° to 80° C, care being 
taken to avoid contact with metallic surfaces. This compound 
should be kept in tightly stoppered bottles in a dark place, as 
oxidation gradually occurs. 

The solution, as used in the test, is as follows: Mix a 
slight excess of phenolphthalin, prepared as above, with 1 
cubic centimeter of — - sodium hydrate solution and a few 
cubic centimeters of redistilled (from glass) water, shake 
thoroughly and filter. To the filtrate add 20 cubic centimeters 



BLOOD IN THE STOOL. 259 

of -^ NaOH solution, 0.1 cubic centimeter of 3 per cent, 
hydrogen peroxid solution, and make the mixture up to 100 
cubic centimeters. This solution should show no trace of pink 
coloration when fresh, but gradually acquires a color, which may 
become so intense that the reagent cannot be employed. In 
forensic work use only freshly prepared solutions. 

To 1 part of the aqueous solution of the stain or of the 
secretion or excretion to be tested add 2 parts of the reagent and 
allow to stand for a few minutes. In the presence of blood a 
pink-red color appears, the intensity depending on the amount 
of blood present. This reaction is retarded by the extracts of 
various animal tissues or various secretions of the body. For this 
reason we are never able to detect as easily small amounts of 
blood in the secretions as in watery solutions of pure blood, the 
limit of delicacy of all reactions being far less in the former than 
in the latter case. Bioiling the solutions before applying the test 
removed most of the interfering factors. If the secretion be 
treated with a thick cream of aluminum-hydrate suspension, the 
precipitate will carry down the blood-pigment and thus concen- 
trate it. A small amount of this precipitate, whether derived 
from saliva, urine, feces suspension, milk, or exudates, will 
show a decided red color when added to 2 cubic centimeters of 
the reagent. Of course, in applying the test to the aqueous 
solution of the blood stain, no such treatment need be employed. 

Webee's Spectroscopic Test for Blood. — Prepare a 
watery suspension of feces. Extract with ether to remove fat 
and then add to the suspension one-third volume of glacial 
acetic acid and mix. Blood if present is converted by this proc- 
ess into acid hematin. The mixture is then filtered and the 
filtrate extracted with two or three volumes of ether. Blood if 
present will discolor the ether, the shade of brown depending 
upon the amount of blood in the specimen. Spectroscopic 
examination of the ether extract is then made for the bands of 
acid hematin (see Plate III). 

Significance of the Occult Blood. — According to Boas, 
occult blood is constantly present in carcinoma of the gastro- 
intestinal tract; intermittently present in gastric and duodenal 
ulcers; occasionally in stenosis of the pylorus, and is absent in 
gastritis, hyperchlorhydria, and in the gastric neuroses. 



260 THE FECES. 

ScHMiDT^s Test' for Ueobiliit. — A small portion of solid 
or liquid feces is rnbbed up in a mortar with four times its vol- 
ume of concentrated aqueous solution of mercuric chloride. This 
mixture is then set aside in a Petri dish for twenty-four hours. 
All particles stained with urobilin are colored red, while those 
stained with bilirubin will be green. The color change may be 
noted within one hour. When not evident the material may be 
examined microscopically. This will show the color reaction in 
even minute particles. 

Schlesinger's Test for Urobilin. — Dilute and rub up in 
a mortar a small portion of feces. If much fat is present extract 
2 or 3 times with ether to remove it. Treat the suspension with 
acid alcohol (same as used in bacteriologic work) and in a short 
time neutralize the acid with ammonium hydrate. Now add to 
the mixture an equal volume of a saturated alcoholic solution of 
zinc acetate, mix thoroughly and filter. The presence of uro- 
bilin will be shown by a green fluorescence in the filtrate. 

Schmidt's Test for Bilirubin. — Proceed as described in 
Schmidt's test for urobilin. Examination will show green 
stained particles in the presence of bilirubin. 

Gmelin's Test for Bilirubin. — The feces must be ex- 
amined fresh, and the test is only applicable when there is a 
relatively large amount of bilirubin present. 

A watery suspension of feces is made as heretofore des- 
cribed. Filter paper is soaked with the suspension and then 
a drop of yellow nitric acid is allowed to fall upon it. A play of 
colors — yellow, red, violet, indigo, and green denote the presence 
of bilirubin. 

BACTERIA AND PROTOZOA IN THE 
FECES. 

There are many varieties of bacteria in the feces. They 
have been estimated to amount to about one-third of dried feces. 
Some are harmless at all times; others, which are, under ordi- 
nary conditions, harmless, may develop pathogenicity under cer- 
tain circumstances. 

The digestive tract of the human organism is, at birth, sup- 
posed to be free from bacterial life ; almost immediately, however, 
organisms gain entrance and rapidly multiply. There is every 



BACTERIA AND PHOTOZOA IN FECES. 261 

probability that they, by virtue of certain chemical substances, 
which they elaborate, are an aid to the digestive processes. Thus 
we know that certain groups of organisms have the power to 
liquefy gelatin, others to digest proteid, etc.; even the mass of 
the bacteria, by their mere presence, are of benefit to the digestive 
tract, since they furnish bulk to the intestinal contents, and 
so aid in the downward movement of digested or partly digested 
material. 

Bacillus Coli Communis. — This organism is constantly pres- 
ent in the feces, and is normally non-pathogenic. It has, how- 
ever, been found in pure culture in cases of appendicitis, 
empyema of the gall-bladder, pyelitis, and cystitis. 

Morphology. 14 — it is a rod with rounded ends, sometimes 
so short as to appear almost spherical, while again it is seen with 
very much longer threads. It may occur as single cells or joined 
together in pairs, end to end. It is motile, and does not form 
spores. It stains with the ordinary alkaline dyes, and is decol- 
orized by Gram's method. 

Compared to the typhoid bacillus, it will be found to be 
less motile; grows more rapidly on gelatine colonies, and lux- 
uriantly on potato (typhoid growth usually invisible). Colon 
bacillus coagulates milk in incubator from thirty-six to seventy- 
two hours; typhoid does not. Colon bacillus decomposes sugar 
solutions; typhoid does not.i^ 

Bacillus Typhosus. — ^This is a distinctly pathologic bacte- 
rium, and is the specific cause of typhoid fever. Its appearance 
in the stools is scanty, but usually can be isolated by appropriate 
methods during the first few days of the disease. 

Pratt, Peabody, and Longi^ obtained it from the stools of 
only 31 per cent, of febrile cases, and none in twenty-one con- 
valescents examined by them. They state that it occurs most 
in the blood, and that it does not develop in the intestinal con- 
tents except under unusual conditions. The bacillus in the in- 
testines comes chiefly from the gall-bladder; frequently it is 
found in the urine in larger numbers than in the feces, and is 
found in greatest numbers in feces containing blood. 



14 Abbott's "Bacteriology." 

15 For methods of plating, isolating, and culture, see works on bacteriology. 

16 Jour. Amer. Med. Assoc, Sept. 7, 1907. 



262 THE FECES. 

Morphology. — The bacillus is about three times as long 
as it is broad, with rounded ends. Its length may vary greatly, 
but its width remains fairly constant. It is actively motile, and 
Loeffler^s method of staining will show it is possessed of many 
delicate flagelli (see methods of staining). 

Its growth on potato usually is invisible. It does not cause 
coagulation of milk (colon bacillus does). Owing to the tend- 
ency to retraction of the protoplasm from the cell-envelope, 
and the consequent production of vacuoles in the bacilli, the 
staining is often irregular. It stains with the ordinary aniline 
dyes. 

Bacillus lactis aerogenes and the bacillus proteus vulg^aris, 
are probably always present in the stools. They are probably 
pathologic in many cases of cholera infantum. 

Gas Bacillus in Feces. ^"^ 

Technic. — 1. Fill a fermentation-tube and a large test- 
tube with nitric acid. 

2. Pour off acid and rinse with hot water until neutral to 
litmus. 

3. Treat glass spatula in same way. Then place 1 c.c. of 
dextrimaltose and 1 c.c. of stool in % test-tube of water. 

4. Boil vigorously % minute and pour into fermentation- 
tube (see Fig. 56, page 251). 

5. Stopper with flamed cotton and place in incubator for 
24 hours (37°). 

6. After 24 hours inspect the tube. 

Interpretation" oe Eeaction. 

A. Bubble like pinhead negative 

B. % in., of air in tube 1 plus 

C. 1 in. of air in tube 2 plus 

D. 1% in. of air in tube 3 plus 

E. More than this or ^^low out tube'^ 4 plus 

Streptococcus aerogenes. — This is an etiologic factor in 
certain cases of entero-colitis. 



17 Philip H. Sylvester and Freeman H. Hibben, Archives of Pediatrics, 
June, 1915. 



BACTERIA AND PROTOZOA IN FECES. 263 

Bacillus dysentericus (Shiga bacillus) is the cause of one 
form of infantile dysentery, and is generally present in the dis- 
charges of such patients showing blood and mucus. 

The Comma Bacillus. — This is the infective agent in Asiatic 
or true cholera. 

Morphology. — It is a slightly curved rod of a length of 
from one-half to two-thirds that of the tubercle bacillus, but 
thicker. Its curve is never very marked, it may be nearly 
straight. It is a flagellated organism, but has only one flagellum 
attached to one end. It is actively motile. It does not form 
spores. '■ 

CuLTUEAiJ Characteristics. — On gelatine plates at room- 
temperature, its development can be observed after as short a 
period as twelve hours. It rapidly liquefies gelatine. It is 
strictly aerobic. It takes the ordinary stains. 

Diagnostic Method. — A smear is made from one of the 
small, slimy particles found in the semi-fluid evacuations, dried, 
fixed, and stained in the ordinary way. If upon microscopic 
examination only curved rods, or curved rods greatly in excess of 
all other organisms, are found, the diagnosis of Asiatic cholera 
most probably is correct. 

Tubercle Bacillus. — This organism probably is always pres- 
ent in all cases of intestinal tuberculosis. 

Diagnostic Technic (E. C. Eosenberger^s) . — A selection 
of ^%ny part of feces is made, there being no effort at a selection 
of any particular mass or portion. If the stool is solid a small 
mass is mixed with sterile water. After dr}dng and fixing, the 
spread is stained with carbol fuchsin for fifteen or twenty 
minutes, cold. The excess of stain is dried off and Pappen- 
heim's solution (see Appendix) is applied, and when tlie 
preparation is the color of the counter stain (methylene-blue) 
thorough washing in distilled water is resorted to, the spread 
being then dried and mounted in balsam. The most important 
point in the technic is to obtain a spread the color of the counter 
stain with not a particle of the carbol-fuchsin showing to the 
naked eye."!^ 

It is alleged, but not conclusively determined, that no other 

18 Solis-Cohen : New York Med. Jour., Aug. 21, 1907. 



264 THE FECES. 

acid- or alcohol- fast bacilli will withstand this method except- 
ing the tubercle bacillus. 

Ameba Dysenterise. — (This is now generally conceded to be 
the specific cause of the so-called amebic dysenter}^ In typical 
cases the stools contain much blood-stained mucus, containing 
large numbers of these amebse. These parasites so resemble 
epithelial cells that a positive diagnosis can only be reached when 
they are seen under the microscope to move and to extend their 
pseudopods. To keep them alive sufficiently long for examina- 
tion, the feces must be caught in a warm pan and a portion im- 
mediately transferred to a warm microscope slide for examina- 
tion. The organism is, from 8 to 50 microns in diameter (see 
Plate VI). To develop the nucleus the organisms should be 
obtained fresh; they are killed by the addition of a few drops 
of acetic acid or corrosive sublimate (sat. watery solution) while 
on the microscope stage. The nucleus is spherical and about 5 
microns in diameter (see page 180). 

For intestinal worms, ova, etc., see section on '^Animal 
Parasites.'^ 

THE CLINICAL SIGNIFICANCE OF THE 
EXAMINATIONS. 

Mucus. — ^As a rule, the appearance of mucus in the stool 
indicates the presence of inflammation of the mucous membrane, 
and is the one trustworthy sign of that condition (Steele). 
There are two conditions in which mucus has no significance: 

1. When thin mucus spreads over the surface of a hard stool. 

2. The so-called mucus-colitis with the discharge of mucus casts. 

Bile-pigment. — ^A green color of part or all the stool (by 
the sublimate test) is pathologic, except in children. It means 
too short a period of passage through the intestines. A fresh 
normal stool should give a pink reaction with IIgCl2. If a color 
reaction of any kind is absent it indicates either absence of 
bile from the intestine, a very fatty stool, or the reduction of 
hydrobilirubin (urobilin) into leucohydrobilirubin, a colorless 
product (Steele). 

Tat. — It will require some practice in the use of the diet to 
tell whether there is an excess of fat in the stool or not. As the 



FOKEIGN BODIES, CALCULI, CONCRETIONS. 265 

normal amount of fat varies within wide limits in normal feces, 
only a great excess can be considered abnormal. 

Remnants of Meat. — Normally there should be only micro- 
scopic particles of connective tissue and muscle fiber. An excess 
of either is often visible to the naked eye. 

Excess of connective tissue means insufficient gastric diges- 
tion, because fibrous tissue^ is digested in the juices of the stom- 
ach only. Excess of undigested muscle-fiber means disturbance 
in intestinal digestion, and probably means trouble in the upper 
part of the small intestine, as follows: (a) Indicating a total 
lesion of the pancreas and absence of proteolytic and tryptic 
digestion. (&) Trypsin may be present, but its activating en- 
zyme, enterokinase, may be absent, (c) The period of passage 
of the food through the intestine may be so rapid that no time 
is given for its digestion, (d) Large pieces of undigested mus- 
cle may be present, because gastric digestion is imperfect and 
the connective-tissue framework of the meat has not been prop- 
erly removed. 

Pathologic carbohydrate fermentation means poor starch 
digestion and indicates, as a rule, disturbance in the small intes- 
tine, which is usually due to insufficiency in the enter icus. 

Pathologic albumin fermentation means a large remainder 
of albumin in the feces, and indicates, in Schmidt's experience, 
serious trouble, usually some anatomic change in the mucous 
membrane of the small intestine (Steele). 

FOREIGN BODIES, CALCULI, AND CONCRETIONS. 

An attack of biliary colic or gall-stones may be followed by 
the appearance, from time to time, of gall-stones in the feces, 
or they may appear without the previous occurrence of any 
symptoms referable to the liver or its appendages. In search- 
ing for them, the feces should be rendered thoroughly fluid by 
rubbing in water and then by passage through a sieve. Various 
forms of simple and complicated apparatus are upon tlie market, 
and are known as stool-sieves. A very satisfactory home-made 
substitute is made by filling in, a wire-frame with two or three 
layers of gauze or cheese-cloth, the wire-frame being arranged to 
fit around the rim of the bowl of the closet. To be certain of 



266 THE FECES. 

not missing the stones it is necessary to examine all movements 
for at least fourteen days following the cessation of the attack 
of colic. Even a careful and prolonged search may fail to find 
the concretion in certain undoubted cases of gall-stone disease. 
Sometimes because the stone causing the symptoms was not 
passed^ but after being jammed in the neck of the bladder finally 
returned into the cavit}^ of the gall-bladder itself. Again, the 
stone may be retained for a long time in a fold or diverticulum 
of the intestinal tract, and finally the concretion may have fallen 
to pieces in the intestine. Another explanation of why the stones 
fail to appear is that t}'pical attacks of colic may be du^ to in- 
flammation without the presence of gall-stones. 

Gall-stoxes are concretions which form in the biliary pas- 
sages. They vary in size from ai pin-head to that of a pigeon^s 
egg or even larger. They are composed chiefly of cholesterin 
and calcium-bilirubin in varying proportions; besides these 
there are present minute amounts of other Inle-oxidation prod- 
ucts and calcium-carbonate. A predominance of cholesterin 
produces a light-, and of calcium-bilirubin a dark-colored stone, 
the absolute color varying usually between a light- or dark- 
brown to a dark-olive green. Stones vary greatly in hardness, 
and on cross-section usually show distinct concentric layers of 
crystalline substance, sometimes of different colors. The sur- 
faces may present a beautiful faceted formation from attrition 
between a number of stones lying 'in the bladder, or they may 
be irregularly granular. 

It is important not to confound other intestinal concretions 
with gall-stones. Woody bits of food, particularly the cores of 
pears, have been termed pseudogall-stones. The microscopic 
examination of bits of these scraped off with a knife, present the 
picture of characteristic wood cells. Chemical examination will 
also prevent mistake (see below). 

So-called biliary sand, in most instances, consists of these 
small pseudogall-stones. The existence of true biliary sand has 
not yet been conclusively proven. Another kind of pseudogall- 
stone consists of balls of fat and fatty soaps which are not easily 
melted. They are found in the stools after the administration 
of large amounts of olive oil, as in a favorite method of treating 
cholelithiasis. 



FOREIGN BODIES, CALCULI, CONCRETIONS. 367 

For chemical examination gall-stones are at first dried, then 
powdered and treated with alcoholic ether to extract the choles- 
terin. This may then easily be recognized by allowing the 
extract to evaporate upon a watch-glass, when the characteristic 
glistening rhomboids of cholesterin crystallize out, and may 
be easily recognized by the microscope. After extraction with 
alcohol and ether the residue is treated, while cold, with very 
dilute potassium hydroxid. If the powder contains calcium- 
bilirubin, a yellow solution will be obtained which gives Gmel- 
lin's reaction. 

The much rarer pancreatic concretions differ from gall- 
stones in that they contain no bile-coloring matter, and are com- 
posed chiefly of calcium carbonate, which dissolves readily with 
effervescence in hydrochloric acid. 

Intestinal Stones or Fecal Concretions. — These are sup- 
posed to play an important part in exciting attacks of appendi- 
citis, but seldom appear in the stools. They consist almost 
exclusively of ammonio-magnesium phosphate (triple phos- 
phate), and should be examined after the manner of urinary 
calculi (see page 364). ^^ 



19 Sahli's "Diagnosis.' 



X. 

THE URINE. 

PART I. 

GENERAL CONSIDERATIONS. 

The normal constituents of the urine are usually tested for 
by quantitative methods, since we are concerned with the actual 
amount of these substances and not with their presence or ab- 
sence from a given sample. In testing for abnormal constitu- 
ents, on the other hand^ we are concerned, as a rule, with their 
presence or absence, though in certain instances we may desire 
to know the absolute quantity of these abnormal substances. 

The Sample. — If we are to make a qualitative examination 
for abnormal constituents in a single sample of urine, it is best 
to collect a specimen about three hours after the ingestion of 
a hearty meal (dinner), as such a sample is most likely to con- 
tain the substances sought (usually albumin or sugar) . 

Cases which at times show a trace of albumin will usually 
show an increase late in the day or after active exercise, and the 
collection of specimens should be timed to meet these conditions. 

Tlie Twenty-four Hours' Specimen.'^ — For accurate results, 
particularly by quantitative methods, it is necessary to examine 
a portion of the mixed urine voided during twent3^-four hours. 



1 Directions for Collecting Urine.— The urine should be coHected in a 
perfectly clean vessel and four ounces sent to the laboratory. Wide-mouthed 
four-ounce bottles especially adapted for this purpose should be obtained at 
the drug stores, and when possible the urine should be directly passed into 
the bottle. 

The evening specimen is to be obtained in the following manner. Empty 
the bladder immediately before the evening meal and discard this urine. 
From the urine first passed after the evening meal, take four ounces and note 
the hour when voided. 

The second specim,en is obtained from the urine first passed upon arising 
in the morning. Note the hour when the urine was passed. 

To oltain the total quuntity of urine passed in 24 hours. On the day when 



(268) 



DECOMPOSITION CHANGES IN NORMAL URINE. 269 

Such a specimen should be examined within six or twelve hours 
after the collection is complete; this will usually prevent errors 
due to the processes of decomposition and putrefaction which 
might destroy the formed elements. 

Catheterized Specimen. — Catheterization is often resorted 
to in order to obtain urine free from contamination, which might 
enter it from the lower part of the urinary tract. For specimens 
of urine from one or both kidneys the ureters may be catheterized. 

Urine kept at room-temperature, particularly in summer 
time, readily undergoes decomposition and putrefaction. These 
changes render it unfit for examination. 

DECOMPOSITION CHANGES IN NORMAL URINE. 

If no method of preservation of the sample is employed, 
the fresh urine, which is clear of acid reaction and showing no 
deposit, will gradually undergo the following changes : — 

1. The lower part grows cloudy from sedimentation of 
mucus, cells and other detritus; the urine is still acid. 

2. This sediment gradually settles to the bottom and may 
show minute crystals of uric acid; the urine is less acid. 

3. Uniformly cloudy from beginning, precipitation of phos- 
phates. The urine is very faintly acid or neutral. 

4. Very turbid from precipitation of phosphates and devel- 
opment of bacteria. Copious sediment of triple and amorphous 
phosphates, bacteria, ammonium urate, and epithelial dchris. 
Alkaline reaction and ammoniacal odor. 



the observation is begun, at a definite hour, empty the bladder, and discard 
this urine. All the urine passed afterward is to be collected in a suitable, 
clean, dust-proof receptacle and kept in a cool place. The following day at 
the same hour, when the bladder is first emptied and the urine discarded, 
again empty the bladder. This urine should be added to complete the total 
ajnount for twenty-four hours, which should be expressed in ounces. After 
the total amount of urine has been collected and thoroughly mixed, send -i 
ounces of the mixture. 

Example. Observation began on January 1st, at 8 A.M. The bladder is 
emptied at 8 a.m.; this urine is discarded; the urine passed during the day and 
night is saved. The next morning, January 2d, at 8 A.M., the bladder is again 
emptied, and this urine is added to comiplete the total quantity for twenty- 
four hours. A label on which is written the name, date, and time when the 
urine is passed should be pasted on the bottle. 



270 THE UIlIISrE. 

PRESERVATION OF SAMPLE. 

When, for any reason, it becomes necessary to delay tlie 
examination of urine past the time when decomposition changes 
nsnally occur, these changes may be retarded in a number of 
ways: 1. By refrigeration. 2. By the addition of toluene 
(toluol) 4 or 5 drops to the ounce. Toluene rarely interferes 
with other urinary tests, its chief objection is that it does not 
mix with the urine but floats on top and so must be wiped from 
the pipette when sediments are being removed for examination 
and the urine syphoned off before commencing chemical tests. 
It successfully inhibits bacterial growth, preserves diacetic acid 
for weeks (Morris) and appears to improve the clearness of 
organized sediments. When not at hand the following sub- 
stances may be used, but it should be remembered that they all 
to some degree interfere with certain reactions. 3. By the addi- 
tion of two or three grains of chloral for each ounce of urine. 
4. By the addition of 10 drops of 4-per-cent. formaldehyde 
solution for each ounce of urine. 5. By shaking with chloro- 
form in the amount of 5 drops to the ounce of urine. 

DESCRIPTION AND IMPORTANCE OF THE URINE. 

The urine is an aqueous solution of the by-products of 
metabolism, so far as these are not excreted or eliminated by 
the lungs, bowels, or skin. It is the most important excretory 
product of the body, and is the medium through which the end- 
products of nitrogenous metabolism and the soluble mineral salts 
are almost exclusively excreted under normal conditions. Ab- 
normal products of metabolism and many substances which have 
found their way into the circulation from without and which 
are foreign to the body, are likewise carried out in solution. 

Not less than 50 per cent, of the total fluid ingested daily 
is excreted as urine. 

General Characteristics. — Normal urine is perfectly trans- 
parent when voided, but soon becomes turbid, and on standing 
deposits a light flocculent sediment composed of a mucinous body 
and a few epithelial cells and leucocytes. If the urine is kept 
cold and care is exercised to exclude the entrance of micro-organ- 
isms, the upper portion of the urine will remain clear indefin- 



PHYSICAL CHARACTERISTICS OF URINE. 271 

itely. Ammoniacal fermentation, due to the activity of the lac- 
terium urea and the micrococcus urea, causes cloudy urine from 
the precipitation of the phosphates. 

Bacteria-free j urine may, in winter, become cloudy because 
the contained urates are less soluble in cold urine than in warm 
urine. On standing the urates settle to the bottom, and the 
supernatant liquid remains clear as long as bacterial contamina- 
tion does not occur. 

Passage of Turbid Urine.^ — In man the passage of turbid 
urine is always abnormal, except during the first days of life, 
when the turbidity is due to the profuse desquamation of epithe- 
lial cells and the relatively large amount of urates. 

PHYSICAL CHARACTERISTICS OF THE URINE. 

The Color. — The color of urine normally varies from a light 
yellow to a dark amber. This is largely influenced by the con- 
centration of the secretion and by the reaction. The pigmenta- 
tion is due chiefly to the presence of a substance called uro- 
chrome (derived from the biliary pigments) and indoxyl potas- 
sium sulphate (indican). Acid urine is always darker than alka- 
line; the color is naturally lighter when the excretion is 
abundant than when it is scant. 

Pathologic urines are subject to great variation in color 
and may vary from a very pale lemon or straw color to a dark 
opaque brownish black. These dark urines are the result of the 
presence of abnormal products of metabolism or altered blood 
pigments and bile. Less marked variations are noted in normal 
persons, the significance of which is not always easily explained. 

Pale urine may occur as a neurosis or during the course of 
certain nervous diseases^ particularly in epilepsy and hysteria. 
It may be a symptom of chronic nephritis or of diabetes. 

Darh urine, which is clear^ occurs in the course of most 
acute fevers, and is due to the presence of uro-erythrin. K 
smoky color denotes the presence of decomposed blood, as in 
acute nephritis. Blood-red or pink urine usually denotes the 
presence of fresh blood. 

Bar and Duaney^ have called attention to a false bloody 

2 Le Progres Medicale, Mar. 24, 190G. 



272 ' ' THE URINE. 

urine due to the activities of a pseudomembranous and chromo- 
genic bacterium. 

Yellow-hroivn or greenish urine suggests the presence of 
bile. Brownish urine occurs in melanosis and after the inges- 
tion of rhubarb, senna, or tannic acid. 

Smolcy-hrown urine indicates the presence of the end-prod- 
ucts of ingested carbolic acid or its analogues. 

Pale greenish urine, with a high specific gravity, usually 
indicates glucose. 

White urine denotes the presence of pus or chyle. 

Whitish turbidity denotes : 1. Pus. 2. Phosphates. 3. Am- 
monium urate. 

Medicinal Discoloeation^ of Urine. — Methylene blue 
produces a greenish or deep blue discoloration which is in- 
creased by the addition of acetic acid. 

Sulphonal, trional, antipyrin, etc., occasions a deep wine 
color due to formation of hematoporphyrin. 

Pyramidon discolors the urine rose-red. This pigment is 
soluble in ether and chloroform. 

The pigments of beets, blackberries and huckleberries may 
under certain conditions be excreted in the'urine as shown by a 
red discoloration. 

Urine v^hich Darkens on Standing. — Some urine, after 
standing for a variable period, becomes dark-brown or black. 
This may be due to the presence in the specimen of melanin or 
of phenol. Non-pathologic phenol urine or ^''carbol urine" may 
arise from medicinal or surgical treatment with phenol or closely 
allied compounds. Under these conditions the end-products in 
the urine usually appear as resorcin. Such urine, if alkaline 
when voided, rapidly darkens on exposure to the air, while if acid 
it becomes dark more slowly, as the alkaline reaction gradually 
develops. 

Under certain pathologic conditions the urine may darken 
on exposure to the air, owing to the presence of alkapton. This 
condition is known as alJcaptonuria. A similar darkening in 
pathologic urine may result from the presence of a coloring 
matter termed melanogen. 

Test. — To differentiate between the phenols and melan- 
ogen: Add bromine water to the suspected urine. In the 



THE AMOUNT. 373 

presence of phenols there will be produced a permanent, yel- 
low precipitate. A primary yellow precipitate which gradually 
darkens, becoming finally brown or blacky indicates melanogen. 

Corrohorative Test. — Add to the fresh undarkened urine a 
few drops of dilute solution of neutral ferric chloride. A violet 
discoloration denotes phenols, brown or black denotes melan- 
ogen. 

It must be remembered that urine containing alkapton will 
give many of the chemical tests for sugar which depend on a 
reduction process. 

The Odor. — Fteshly passed normal urine has a peculiar 
characteristic aromatic odor, resulting from the contained 
volatile acids. Decomposing urine has the characteristic odor 
due in part to the free ammonia resulting from the decomposi- 
tion of urea. 

Urine which is ammoniacal when freshly passed, points to 
a pathologic fermentation occurring in the bladder, usually ac- 
companying cystitis. 

A PUTRID odor denotes putrefactive change occurring in pus 
or other albuminous substances. 

An odor resembling acetone is occasionally observed and 
denotes diabetes mellitus. 

Urine containing cystin may, upon standing, develop the 
odor of HYDROGEii;r sulphide. 

Some articles of food, as asparagus and onions, and certain 
aromatic medicines, as turpentine and copaiba, may give char- 
acteristic odors. 

THE AMOUNT. 

The average daily excretion of urine in the United States 
is somewhat less than in foreign beer-drinking countries. A 
fairly normal average for the United States may be stated to be 
between 1200 and 1600 cubic centimeters, or about 50 ounces 
for men, while for women it is slightly less. Generally speaking, 
the amount will vary in inverse ratio to the insensible perspira- 
tion: hot weather diminishes and cold weather increases tlie 
amount. Profuse perspiration, sweating, vomiting, and diar- 
rhea all decidedly diminish the amount. Children, and espe- 
cially nursing infants, on account of the preponderance of liquid 

IS 



274 THE URINE. 

in their food^ excrete a proportionately larger amount of urine, 
compared with body-weight, than do adults. 

The normal amount of urine passed in twenty-four hours is 
consequently subject to wide variations, depending on the amount 
of fluid ingested, the character and the quantity of the food, 
the process of digestion, the blood-pressure, the surrounding 
temperature, the emotions, sleep, exercise, age, sex, and body- 
weight. During repose much less urine is excreted than during 
activity, hence the excretion during the night is less than during 
the day. The maximum secretion is usually observed during the 
first few hours succeeding a hearty meal. 

Artificially the excretion may be increased by substances 
which have a tendency to raise blood-pressure, as tea, coffee, 
and alcohol. Many drugs bring about the same result. The 
most important of the medicinal diuretics are digitalis^ squill, 
broom, juniper, nitrous ether, urea, etc. Distilled water also 
possesses distinct diuretic properties. 

The quantity of urine in pathologic conditions depends, first 
on the condition of the secreting renal parenchyma and, sec- 
ondly, upon the condition of the blood-current in the kidneys. 
It will therefore be affected in general by circulatory disturb- 
ances, as well as by disease of the kidney itself. To appre- 
ciably alter the amount of the excretion, both kidneys must be 
diseased, for if one kidney be healthy it will assume vicariously 
the function of the other organ. This compensatory action 
regularly occurs after extirpation of one kidney. 

As a rule the more acute the nephritis the more the excre- 
tion of urine will fall below normal, while the more chronic the 
process the more will the amount exceed normal. This increase 
reaches its maximum in the true contracted kidney where the 
volume excreted in twenty-four hours may be very great (from 
2000 to 4000 cubic centimeters). Diseases of the heart and 
lungs, leading to chronic passive congestion, will diminish the 
total amount of the urine, a condition evidently dependent upon 
interference in the renal circulation. 

It is evident, therefore, that the determination of the total 
output of the kidneys and an observation of the variation in the 
amount during tlie course of disease, may be of great diagnostic 
and prognostic value. 



SPECIFIC GRAVITY. 275 

The increased excretion of urine following convulsions, par- 
ticularly the hysterical variety and after attacks of angina pec- 
toris, is probably dependent upon some vasomotor disturbance. 
Many alterations in the volume of the urine depend upon quan- 
titative and qualitative variations in the substances eliminated, 
and are primarily induced by disturbances in metabolism. 

Diurnal Variation. — Chronic contracted kidneys tend to 
accumulate urine in the body during the day and to excrete it 
during the night. This often producing a reversal of the nor- 
mal relations betv^een the day and the night urine. 

Anuria, or entire failure to void urine, may be due either 
to complete suppression of the secretion in the kidneys or to 
obstructions in the urinary tract. 

Hydruria. — This is a state of the urine in which the fluid 
is increased out of proportion to the solids, and is usually asso- 
ciated with an increase in the total twenty-four hours' output. 

Polyuria. — This term is applied to an increase in the elimi- 
nation of the urine as a whole, both fluids and solids. 

Oliguria is applied to a diminution in the total excretion 
of the urine. 

Polyuria may be noted in the following conditions : 1. Dia- 
betes mellitus. 2. Diabetes insipidus. 3. Chronic interstitial 
nephritis. 4. Amyloid disease of the kidney. 

Oliguria is met under the following circumstances: 1. 
Valvular heart-disease. 2. Diminished blood-pressure (see sec- 
tion on blood-pressure). 3. Acute articular rheumatism. 4. 
Chronic parenchymatous nephritis. 5. Acute congestion and 
inflammation of the kidneys. 6. Decreased cell activity in 
shock. 7. Failure of nutrition preceding death. 

SPECIFIC GRAVITY. 

The average specific gravity of normal urine of 1500 centi- 
meters (fifty ounces) volume for twenty-four hours, is about 
1020. Slight variations (1015 to 1028) from this standard are 
consistent with perfect health and depend chiefly upon the char- 
acter of the food ingested, the quantity of water taken, and upon 
the state of metabolism. Under certain conditions of apparent 
health, as after a hearty meal, the specific gravity of the indi- 



276 THE URINE. 

vidual specimen may be as high as 1035, and after excessive in- 
gestion of fluids it may temporarily fall to 1005. 

Under pathologic conditions the specific gravity may vary 
between 1001 and 1055 or even higher. If the diet consists 
largely of nitrogenous food it will furnish a relatively larger 
amount of solids; in consequence the specific gravity of the 
urine will be increased. Active muscular exertion also tends to 
raise the specific gravity by increasing tissue catabolism. Co- 
pious diaphoresis may bring about a concentration of the urine 
by diminishing the amount. Fasting has a similar effect. 

The specific gravity usually varies inversely with the volume 
both under normal and pathologic condition. A scanty urine is 
more concentrated than a profuse one. Diabetes mellitus forms 
an exception to this rule, since the presence of sugar produces 
a high specific gravity in spite of the excessive amount of the 
excretion. This fact is so characteristic that a tentative diag- 
nosis may be made upon this alone. 

From the foregoing it is evident that, from a clinical stand- 
point, it is necessary to consider the specific gravity, the total 
solids, and the total volume together, as they are intimately and 
logically related to each other, and that the determination of 
the specific gravity is of greatest value and significance when 
the total volume of the urine for twenty-four hours is known. 
Also that the chief clinical value of the study of the amount and 
of the specific gravity is to aid in the estimation of the total 
solids of the urine (see below). 

The specific gravity of the urine is usually determined with 
the aid of a hydrometer or urinometer, but is more accurately 
determined by the Westphal balance. Only approximately cor- 
rect results may be obtained with the urinometer. 

The Use of the Urinometer. — The scale of the urinometer 
is usually marked in regular intervals from 1000 to 1060 (Fig. 
57). To insure ease and accuracy in reading, these markings 
should not be too close together. Many urinometers are 
inaccurately made, so that before purchasing an instrument it 
is always well to compare it with a standard instrument, or at 
least to ascertain that it floats at the 1000 mark in distilled water 
at the standard temperature (15° C. or 60° F.). Although a 
large instrument is more accurate, a small one, requiring less 



SPECIFIC GRAVITY. 



27? 



nrine, is more convenient and must frequently be employed 
for lack of sufficient urine in which to float the larger instru- 
ment. 

To determine the specific gravity of the urine : After allow- 
ing the urine to cool to room temperature (about 60° ¥.), it is 
poured into the urinometer tube (the urinometer and an appro- 
priately sized glass cylinder are usually sold together), and the 
urinometer immersed in the urine ; then with the eye' on a level 
with the surface of the urine, the division of the scale is read 
off which corresponds to the lowest part of the curve of the 




Fig. 57.~Ukinometer and Cylinder. 

meniscus. To insure accuracy the containing cylinder should 
be sufficiently large to allow the urinometer to float freely and 
not come in contact with the sides. All bubbles and froth should 
be removed from the surface of the fluid by means of filter-paper. 
If the urine is cooler than 15° C, one-third of a urinometer 
unit should be subtracted for every degree centigrade below the 
standard temperature. If warmer an appropriate addition 
should be made. Since the specific gravity of individual urine 
specimens vary greatly during twenty-four hours, it is neces- 
sary that the specific gravity should be taken from a mi.\ed 
specimen of the twenty-four hours' collection. A reading taken 
from any single specimen is of little clinical significance. 

If the specimen is too small to work with in the ordinary 
way, it is a very simple matter to dilute with a known proper- 



278 THE URINE. 

tion of distilled water. Estimate the specific gravity of the 
diluted -urine and then calculate the specific gravity of the speci- 
men at least approximately. 

The Westphal balance is an extremely accurate method of 
determining the specific gravity, carrying the reading to the 
fifth figure (fourth decimal). The method of employing the 
Westphal balance is as follows : — 

When the instrument is mounted the glass plummet which 
is suspended from one end of the beam will balance in distilled 
water at 15° C. and represents 1. Now pour the urine into the 
jar until the twist in the platinum wire is below the surface. 
Weighing is accomplished as follows: Place the first rider upon 
the end of the beam above the float, then place the second rider 
in the first notch to the left on the scale on the beam. If the 
plummet rises, place the rider on the second notch. If now the 
beam balances and the temperature of the urine is 15° C, the 
specific gravity of the urine is exactly 1020. If the float still 
rises, take the third rider and find the notch in which the beam 
balances or nearly so. If the beam balances with the third rider 
in the fourth notch, the specific gravity is exactly 1024. Should, 
liowever, the float still slightly rise, take the fourth rider and 
find the exact balance, and if this is in the sixth notch the 
specific gravity is exactly 10246. In other words, the second rider 
(in size) gives the third figure, the third rider the fourth (third 
decimal), and the fourth rider the fifth figxire or the fourth 
decimal. With a little practice determinations with the West- 
phal balance are rapidly and accurately made. Care should be 
exercised to see that no air bubbles become attached to the cord 
or float, and that temperature corrections are made as directed 
below. 

Corrections for Temperature. — The temperature of the 
urine immediately after being voided ranges from 85° to 95° 
F. (29.5° to 35° C.) ; therefore in taking the specific gravity 
of fresh urine its temperature must be observed, and for every 
seven degrees Fahrenheit that the thermometer indicates above 
the temperature at which the instrument is standardized, one 
degree should be added to the specific gravity, as indicated by 
the instrument. 



THE REACTION. 279 

METHOD OF ESTIMATING THE TOTAL SOLIDS. 

The accurate chemical method of weighing is too difficult 
and tedious for general clinical work, descriptions of which may 
be found in works on physiologic chemistry. . 

The normal average weight of solids is 65 grams. The 
specific gravity is a pretty accurate index of the amount of 
solids excreted by the urine when this has been determined from 
the twenty-four hours' urine. 

The solids excreted in 1 liter of urine may be approxi- 
mated in grains by multiplying the last two figures of the spe- 
cific gravity by 2.2337 (Vierodt's factor). 

Trapp's Formula. — Multiply the last two figures of the 
specific gravity by two, and the result will represent the parts 
of solids in 1 liter of urine. Example: If the specific gravity 
is 1023, then 23 times 2 will equal 46 parts of solids per 1000 
cubic centimeters. 

Bird's Formula. — ^The last two figures of the specific gravity 
about represent the grains of solids in a fluidounce of urine 
tested. Thus, a specific gravity of 1022 would contain approxi- 
mately 22 grains of solids per ounce of urine. 

Metz's Formula. — ^Multiply the last two figures of the specific 
gravity by 0.00233, and multiply this product by the total twenty- 
four hours' volume in cubic centimeters. The final product will 
be the total weight of solids expressed in grams. Example: 
Specific gravity = 1024; then 24x0.00233x1500 cubic centi- 
meters equals 87.27 grams of solids in twenty-four hours' 
excretion. 

THE REACTION. 

The reaction of the twenty-four hours' urine is usually acid, 
sometimes amphoteric, and rarely alkaline. The normal acidity 
is not due to the presence of free acids, but to acid salts, chiefly 
acid phosphates. Primarily the reaction of the urine depends 
upon the character of the diet. Increase in ingestion of food- 
stuffs containing alkaline salts (vegetables), or when such salts 
are formed within the body from organic acids contained in the 
food (fruits), will tend toward an alkaline reaction when the 
urine is voided. The elimination of alkaline urine in this in- 



280 THE URINE. 

stance is due to the presence of a fixed alkaline, and therefore 
cannot be considered pathologic. 

It has already been stated that the acid reaction of urine is 
normally due chiefly to the acid phosphates. Besides these a 
certain amount of the neutral phosphates of calcium, ammonium, 
and of sodium usually occur, and it may happen that the acid 
and neutral phosphates are present in such proportions that the 
sample will turn blue litmus red and red litmus blue. Such a 
urine is said to be amphoteric. 

Urine, when allowed to remain exposed to the air at room- 
temperature, gradually undergoes ammoniacal fermentation. 
This is due chiefly to the action of certain micro-organisms upon 
urea, which is decomposed by a hydrolytic process into ammonia, 
carbon dioxid, and water (see page 269). 

The effect of this development of an alkaline reaction is 
precipitation of the soluble phosphates of the alkaline earths, as 
tri-calcium phosphate and ammonio-magnesium phosphate; at 
the same time the soluble urates are transformed into the 
insoluble ammonium salt (see Fig. 60). 

Test. — In order to determine whether the alkalinity of a 
given specimen is due to a fixed or to a volatile alkali (am- 
monia), a strip of red litmus is clamped into the cork of the 
bottle so that it does not touch the liquid but remains dry. 
Volatile alkali will gradually turn the paper blue, while a fixed 
alkali will not affect the paper unless the alkaline fluid touches it. 

In specimens where the degree of alkalinity or acidity is so 
slight that the litmus-paper test leaves the examiner in doubt, 
the uncertainty may be eliminated by the following expedient: 
Take two pieces of litmus-paper, one red and the other blue; 
allow the urine to come in contact with but half of each strip 
of paper; then with distilled water allow the whole extent of 
both strips to become wet. This eliminates any change in the 
paper due to the presence of moisture alone, and brings out 
sharply slight changes in the color of the litmus-paper by 
allowing comparison between the adjacent portions of each strip 
of paper. 



DETEEMINATION OF ACIDITY. ggl 

DETERMINATION OF TOTAL ACIDITY 
OF THE URINE. 

Accurate determination of urinary acidity is a difficult and 
tedious procedure, requiring considerable technical skill. For 
general clinical purposes Folin's method is practical, and has the 
advantage of being rapid and simple. It is important to begin 
with a perfectly fresh sample, in order to exclude change in re- 
action due to fermentation and decomposition (see page 269). 
Proteid, if present, must be removed by heat and acetic acid, in 
which case a known amount of acetic acid must be employed 
and this taken into consideration in the titration. 

FOLIN'S METHOD FOR TOTAL ACIDITY. 

Twentj^-five cubic centimeters of urine are treated with 15 
to 20 grams of powdered potassium oxalate and one or two drops 
of a 1 per cent, alcoholic solution of phenolphthalein. The mix- 
ture is shaken rapidly for one or two minutes and titrated at 
once with a tenth-normal sodium hydrate solution (for prepara- 
tion of normal and decinormal solutions see Appendix, page 489) . 
until a faint, distinct, permanent pink color is obtained. It is 
advisable to shake the flask during the titration so as to prolong 
the effects of the potassium oxalate. The acidity is expressed 
in terms of the amount of tenth-normal sodium hydrate solution 
necessary for neutralization of the twenty-four-hour amount of 
urine. This is expressed as T, which is, on an average, 617. 

If the urine to be tested is alkaline, then first add to the 
100 cubic centimeters exactly 20 cubic centimeters of the one- 
tenth normal HCl solution. Stir well, and then proceed as in 
case of the acid urine, deducting the number of cubic centimeters 
^ NaOH from the 20 cubic centimeters ~ HCl used and then 
calculating as T the total neutralization of the twenty-four-hour 
specimen. 

Significance : The acidity in cardiovascular-renal disease is 
usually high. Generally speaking, most pathological condi- 
tions are accompanied by an increase in urinary acidity. 

Increased Acidity. — ^The urine is frequently found hyper- 
acid in: — 

1. Fevers, 



282 THE URINE. 

2. Inflammations of the liver. 

3. HypereHorhydria. 

4. Acute rheumatism. 

5. Lithemia. 

6. Neurasthenia. 

Alkaline urine may be passed under the following condi- 
tions : — 

1. Undue retention of urine in the bladder. 

2. When there is residual urine. 

3. Presence of urea-decomposing bacteria in the bladder. 

4. Chlorosis. 

5. Organic nervous diseases. 

6. In marked degrees of general debility. 

CHEMICAL COMPOSITION OF THE URINE. 

A general idea of the chemical composition of the urine and 
of the quantitative variation of the individual components may 
be had from the accompanying table (after Simon). The indi- 
viduals from whom the urine was obtained were adults. Their 
habits, diet, etc., may be taken to be that of the average Ameri- 
can city dweller. 

It must be borne in mind that tables of chemical composi- 
tion are based upon averages taken from a large number of 
complete analyses, the composition of which, even in perfect 
health, covers a greater range of variation than indicated in the 
table on following page. 

THE INORGANIC CONSTITUENTS OF 
THE URINE 

The inorganic constituents of the urine represent the excess 
of mineral salts that find their way into the blood from the 
digestive tract or which develop within the body during the 
processes of metabolism, especially during albumin decomposi- 
tion. We therefore find that exercise and the ingestion of large 
amounts of food, as well as the increased cell activity occurring 
in acute fevers, lead to an increased elimination of salts, and 
conversely smaller amounts of salts are eliminated when the 



INORGANIC CONSTITUENTS. 283 



CHEMICAL ANALYSIS OF THE URINE (Simon). 

Grams. 

Water 1200 to 1700 

Solids 60 

Inorganic solids 25.0 to 26.0 

Sulphuric acid (H2SO4) 2.0 to 2.5 

Phosphoric acid (P2O5) .... 2.5 to 3.5 

Chlorine (NaCl) 10.0 to 15.0 

Potassium (K2O) 3.3 

Calcium ( CaO) 0.2 to 0.4 

Magnesium ( MgO ) 0.5 

Ammonia (.NH3) 0.7 

Fluorids, nitrates, etc 0.2 

Organic solids 20.0 to 35.0 

Urea 20.0 to 30.0 

Uric acid 0.2 to 1.0 

Xanthin bases 1.0 

Creatinin 0.05 to 0.06 

Oxalic acid 0.05 

Conjugate sulphates 0.12 to 0.25 

Hippuric acid 0.65 to 0.7 

Volatile tatty acids 0.05 

Other organic solids 2.5 



intake of food in general is restricted. These statements apply 
particularly to the phosphates. 

The bases which are found in the urine in combination with 
hydrochloric acid, phosphoric acid, and sulphuric acid, are chiefly 
sodium, potassium, calcium, magnesium, and ammonium. It is 
believed that the mono-acid phosphates of the alkaline earths 
are held in solution by sodium chlorid, and also by the di-acid 
sodium phosphates, to which latter the acidity of the urine is 
largely due. 

While the greater portion of the sulphuric which results 
from albumin decomposition is found in the urine combined with 
inorganic bases, a variable fraction, also occurs united with cer- 
tai^ aromatic substances which are developed in the intestines 
during putrefaction and decomposition. The resulting bodies 
are spoken of as the ethereal or conjugate sulphates. They com- 
prise the alkaline salts of indol, skatol, and phenol. 

The Phosphates. — The amount of phosphoric acid excreted 
by the healthy individual in twenty-four hours ranges from ?.3 
to 3.5 grams, the average being 2.8. In the urine the phosphoric 
acid is found in part combined with the alkaline earths — earthy 



284 THE URINE. 

phosphates — and in part with the alkalies as the alkaline phos- 
phates. The alkaline bases represent about t^vo-thirds of the 
total phosphoric acid in combination. The earthy phosphates are 
insoluble in water^ but are soluble in dilute acids. They comprise 
the phosphates of calcium and magnesimn. The calcium phos- 
phates predominate. In acid urine the earthy phosphates are in 
solution, while in alkaline urine they are precipitated. They are 
thrown down by heat, and also when the reaction of the urine 
becomes alkaline, whether from a course of internal alkaline 
medication or from the putrefaction of the urea of the urine. 
If, during decomposition of the urine, the contained acid phos- 
phates are acted upon by the ammonia resulting from urea 
decomposition, ammonium-magnesium phosphate (triple phos- 
phate) is formed and appears in the urine as the characteristic 
prismatic or coffin-lid crystals. 

The alkaline phosphates comprise the phosphates of sodium 
and potassium; of these the sodium is the more abundant. 
These, unlike the earthy phosphates, are easily soluble in water 
and in alkaline fluids. The alkaline phosphates form the 
chief bulk of urinary phosphates. The normal urinary acidity 
depends upon the presence of these acid phosphates, and not 
upon the presence of free acid. 

"While the bulk of the phosphates in the urine is derived 
from the decomposition of food-stuffs, a part also is derived 
from the breaking down of the highly complex organic bodies: 
lecithin and nuclein. 

Clinicalh^, the excretion of phosphoric acid and its determi- 
nation is of very little significance, since it is so largely de- 
pendent upon the influence of diet, exercise, etc. 

DetectioisT. — If neutral or alkaline urine is heated in a 
test-tube, a precipitate will be formed which will be found to 
consist of earth}' phosphates. Such a precipitate might be caused 
by the presence of albumin. To remove the doubt add a few 
drops of dilute (10 per cent.) acetic acid, when a precipitate due 
the phosphates will immediately disappear, while if albuminous 
the cloud will remain or may be increased. 

Test for Earthy Phosphates. — To a few cubic centim- 
eters of urine in a test-tube add a few drops of liquor potassii 
and boil. The earthy phosphates will be thrown out of solu- 



INORGANIC CONSTITUENTS. 285 

tion^ and may, after settling, be collected upon a filter. Now, 
to the filtrate add one-third of its volume of "magnesia mix- 
ture'^ (for formula see Appendix). The precipitate thus 
formed represents the phosphoric acid that was in combination 
with the alkaline bases, combined now in the form of ammonium- 
magnesium phosphate. 

Estij\iatioi^ of the Phosphates by the Centrifuge. — 
In a graduated percentage centrifuge tube (see Fig. 68) mix 10 
cubic centimeters of urine with 5 cubic centimeters of "magnesia 
mixture"; invert several times to thoroughly mingle. Eevolve 
in the centrifuge for three minutes. Eead off every one- tenth 
cubic centimeter of precipitate as 1 per cent, by bulk of total 
phosphates. The average normal percentage by this method is 
eight. Eoughly, each one-tenth cubic centimeter of sediment is 
equal to about 0.0225 per cent, by weight of P2O5. 

Determination of the Total Phosphoric Acid. — For 
this determination the following solutions are required: — ■ 

1. A standard solution of uranium nitrate : 20.3 grams of 
uranium^ nitrate are dissolved in 1000 cubic centimeters of dis- 
tilled water, then each cubic centimeter of this mixture is 
equivalent to 5 milligrams of phosphoric acid. 

2. Sodium acetate solution : 100 grams of sodium acetate 
are dissolved in 900 cubic centimeters of distilled water^ and to 
this 100 cubic centimeters of acetic acid are added. 

3. Saturated solution of potassium ferrocyanide. 
Method. — Fifty cubic centimeters of urine are placed m a 

beaker and 5 cubic centimeters of the sodium' acetate solution 
added. The mixture is warmed over a water-bath and uranium 
nitrate solution added from a burette as long as a precipitate 
is formed. If the formation of precipitate is not easily recog- 
nized, a drop of the potassium ferrocyanide solution may be 
added; then, as long as a brown color does not appear where 
the drop falls, the uranium nitrate solution should continue to 
be added. The end point in the precipitate reaction is 
reached when a reddish-brown discoloration appears upon the 
addition of a drop of the uranium nitrate. The quantity of 
uranium nitrate solution employed to accomplish complete pre- 
cipitation is now read off from the scale on the burette, each 
cubic centimeter of which will equal 5 milligrams of phosphoric 



286 THE URINE. 

acid. This number and the 50 cubic centimeters of urine em- 
ployed furnished the working bases for calculating the per- 
centage. 

The presence of sugar or albumin does not interfere with 
this reaction. 

Separate Estimation" of the Earthy and Alealine 
Phosphates. — Two hundred cubic centimeters of urine in a 
beaker are rendered alkaline by the addition of ammonium 
hydroxid, and set aside for a few hours. The earthy phos- 
phates are thus precipitated and may be collected upon a filter- 
paper, and after washing with dilute ammonia (1:3) are trans- 
ferred to a beaker, where they are dissolved with as little acetic 
acid as possible. Distilled water is then added so as to make 
the total volume approximately 50 cubic centimeters, when the 
solution is boiled and then, titrated as above. In a second por- 
tion of urine the total phosphates are determined as outlined 
above. Then the difference between the two results will repre- 
sent the quantity of phosphoric acid present in combination 
with the alkalies. 

Significance. — As has been shown above, the phosphates are 
dependent upon many uncertain factors which are determined 
with diificulty and are of little clinical significance. When 
in a given case the phosphates are constantly thrown out of 
solution, it may be taken as an indication that the formation of 
gravel or calculus is impending. If the usual signs of ammo- 
niacal fermentation are present, the significance is plain. 

THE SULPHATES. 

General Considerations. — The major portion of the sul- 
phates appearing in the urine are derived from the food, and 
comprise the simple mineral sulphates of sodium and potassium. 
Only a small portion exists in organic combination as the ethe- 
real or conjugate sulphates. 

The three predominating conjugate sulphates are : Phenol 
potassium sulphate, indoxyl potassium sulphate (indican), and 
skatoxyl potassium sulphate. 

The mineral sulphates comprise about nine-tenths of the 
total sulphates in the urine. 



THE SULPHATES. 287 

Test for Mineral or Preformed Sulphates. — Add a few- 
drops of acetic acid (to prevent the formation of barium phos- 
phate) to a test-tube half full of urine. Now, upon the addition 
of a solution of barium chlorid a whitei precipitate of insoluble 
barium sulphate will be formed. This precipitate varies in 
densit}^ from a faint white cloud to a bulk; that gives a thick 
creamy consistence to the whole mixture. One can roughly deter- 
mine by the amount of precipitate as compared with the known 
normal standard, whether the sulphates are increased or not. 

Test for Conjugate or Ethereal Sulphates. — Mix 
equal quantities of alkaline barium chlorid (see Appendix) and 
urine in a test-tube. After allowing a few minutes for the 
precipitate to form the mixture is filtered. This process precipi- 
tates both phosphates and preformed sulphates. The filtrate is 
now acidified with, 5 cubic centimeters of a (one-fifth vol.) HCl 
solution and then boiled for some time. The presence of ethe- 
real sulphates is indicated by a reddish discoloration of the 
fluid which in addition becomes turbid. 

Potassium Indoxyl Sulphate or Indican. — As this substance 
represents the characteristic etheral sulphate, it may be taken 
as an indicator for the^ whole group, and the tests for this sub- 
stance used for estimating the relative amount of the whole 
group of ethereal sulphates in the urine. The several tests for 
indican are based upon the fact that an excess of HCl will 
liberate the indoxyl, which can then, by the addition of an 
oxidizing agent, be converted into indigo-blue, and finally this 
can be recognized in small amounts by extraction from the bulk 
of urine with chloroform. 

Test for Indican (modified Jaffe). — Take 20 cubic centi- 
meters of filtered urine in a test-tube and add 3 or 4 cubic 
centimeters of chloroform and one drop of a 1 per cent, solu- 
tion of potassium chlorate, and finally 20 cubic centimeters of 
HCl. This mixture is to be thoroughly agitated and mixed by 
pouring repeatedly from one test-tube to another. This should 
be repeated at intervals of two or three minutes, covering a 
period of ten minutes. The presence of indican is indicated by 
a blue discoloration of the chloroform. In the presence of a 
large amount of indican the chloroform will appear almost 



288 THE URINE. 

black, while the whole mixture will assume a dusky bluish-red 
color. (For comparative color-scale see Plate VII.) 

While a trace of indican cannot be considered pathologic, 
there are, nevertheless, many specimens, possibly one-third, 
which fail to show any discoloration of the chloroform. 

Caution. — 1. An excess of oxidizing agent, either in volume 
or strength, will prevent a positive reaction through over- 
oxidation of the indoxyl compound. 2. An excess of chlo- 
roform will result in too great dilution of the indigo, causing 
failure to detect traces of indican. 3. A reddish discoloration 
of the chloroform is not due to indican. Such a reaction occurs 
in the urine of patients who are taking iodid, and possibly bro- 
mid. The sim^ultaneous occurrence of a red and blue re- 
action will produce a color bordering on purple. This possible 
source of error may be removed by the addition of a few drops 
of a 10 per cent, solution of sodium thiosulphate, which will 
bleach the pink color due to iodin. 

Obermater's Test. — This test is also a modification of 
Jaffe's test, with certain improvements suggested by Eosen- 
bloom.3 

To nearly a half test-tube full of urine add an equal quan- 
tity of hydrochloric acid containing 4 grams of ferric chlorid to 
the liter. Mix by pouring from one tube to another and allow 
to stand ten minutes. Add about 3 cubic centimeters of chlo- 
roform and again mix by pouring from one tube to another, then 
permit it to stand fifteen to thirty minutes. The supernatant 
fluid above the chloroform is now poured off and the test-tube 
filled with water. This accentuates the blue color due to the 
presence of chloroform. Thymol, if added to the urine as a 
preservative, affects the end action, the thymol giving the urine 
a violet color. 

Should protein matter be present in the urine, it must be re- 
moved by boiling, precipitation with dilute acetic acid, and filter- 
ing. The filtrate is used in testing for the indican. 

Quantitative Determination of Indican. — The principle of 
this test is the same as Jaffe's qualitative test in that the indi- 
can is oxidized to indigo, which in turn is extracted with chloro- 
form, the chloroform evaporated off, the residue of indigo re- 

3 N. y. Med. Jour.. October 25. 1913. 



fe 



a. 



W O 

3<i 



k5H 



fH H W 

■\ rt ^ ^ 
I w ':2 ** 



5^ 



2; 

« c 

Pi 



THE SULPHATES. 289 

maining finally titrated with potassium permanganate. The 
method while of clinical accuracy is not absolute. 

Technic. — Place 50 cubic centimeters of urine in a small 
beaker, add 5 cubic centimeters of basic lead acetate solution 
(lead acetate 18 grams, litharge 11 grams, distilled water to 
make 100 cubic centimeters) mix well and filter. This removes 
the interfering substances from the urine. Transfer 40 cubic 
centimeters of the filtrate to a separating funnel, add 40 cubic 
centimeters of Obermayer's reagent (3 grams ferric chlorid dis- 
solved in 1000 cubic centimeters HCl), add 25 cubic centimeters 
chloroform and after agitation, extract. Eepeat the process with 
additions of chloroform until the chloroform takes up no more 
color. Shake out the combined chloroform extracts with dis- 
tilled water in the separatory funnel. Eepeat 2 or 3 times and 
finally wash with very dilute sodium hydrate and separate, 
and again wash with water. Finally, filter the chloroform ex- 
tracts through dry paper into a small dry flask, distil off the 
chloroform, heat the dried mixture on a water-bath for five to 
eight minutes, then wash the dried residue with water until the 
wash-water is no longer discolored. Add 10 cubic centimeters 
cone. H2SO4 to the residue, heat on water-bath until completely 
dissolved and dilute with distilled water to 100 cubic centimeters. 
The blue solution may then be titrated against a dilute per- 
manganate of potash solution of known strength. Taking as the 
end point the disappearance of all blue color and the substitu- 
tion of a pale yellow; the content of indican in the original 
specimen may then be calculated. (Dilute permanganate = 3 
grams of potassium permanganate dissolved in 1000 cubic 
centimeters distilled water and keepi in colored bottle) . For use 
dilute each 1 cubic centimeter with 39 cubic centimeters of dis- 
tilled water. Such a solution has the value: 1 cubic centim- 
eter of dilute permanganate solution equals approximately 0.15 
milligrams of indigo. 

The blue solution obtained by the above extraction method 
may if desired be compared with a standard solution of indigo. 
(See next page.) 

Four to 20 milligrams of indican are excreted daily by tlie 
normal adult. 

19 



290 THE URINE. 

Approximate Detennination of Indican. — F. C. Askenstedt^ 
recommends the following modification of the Jaffe technic for 
the determination of indican. 

The following solutions are used: — 

A. 0.4 per cent, solution of iron perchlorid in HCl. 

B. A true solution of indigo blue in sulphuric acid. 
This strength is of such a solution that one drop holds exactly 
0.000165 milligram of indigo blue. (This solution keeps well 
in amber-colored bottles.) 

C. An appropriate 1 : 5000 solution of picric acid in alcohol. 

D. Denatured alcohol. 

The test is carried out as follows : To 10 cubic centimeters 
of urine in a test-tube add 10 cubic centimeters of the ferric 
chlorid solution and mix by inverting the tube once; then add 
quickly 8 cubic centimeters of chloroform, and extract the indigo 
in formation by shaking the tube 400 times, holding it in a hori- 
zontal position. After this let the chloroform fall to the bottom 
of the tube, then pour off most of the supernatant fluid, fill the 
tube nearly full with water, invert it a few times to wash the 
chloroform and let this again precipitate in the tube, and pour 
off most of the water. Repeat twice this process of washing, tak- 
ing care that no chloroform escape with the wash-water, and al- 
lowing not more than 2 or 3 cubic centimeters of the last wash- 
water to remain over the chloroform. Now add from 13 to 15 
cubic centimeters of alcohol and mix by shaking. A clear blue 
fluid should result. If hazy, add one or two cubic centimeters 
more of alcohol until the fluid clears up. Compare the color of 
this fluid with an equal quantity of a standard solution of indigo 
blue in the second test-tube by holding the two test-tubes in front 
of a white surface. This standard solution is made by pouring 
into the empty second tube a quantity of water equal to the 
amount of the fluid in the first tube, and then dropping the 
stock solution of indigo blue into the water, inverting the tube 
after each drop, until both solutions have the same amount of 
blue color. If this requires four drops of the stock solution the 
percentage is 0.0004; if five drops, 0.0005; if six drops, 0.0006, 
etc. 



4 N. Y. Med. Jour., October 9, 1909. 



SKATOL. 291 

Suggestions. — Several drops of the picric acid solution may 
be added to the standard solution in the test-tube in order to 
balance an occasional greenish tinge appearing in the indican 
extract. 

Advantages. — Neither albumin nor bile interferes with 
this estimation. Sugar reduces it. To compensate for indican 
lost during the technic, add 20 per cent, of the amount deter- 
mined for the final result. Urine containing more than 0.002 
indican, or which has a blackish extract, should be diluted with 
equal parts of water and retested. The presence of formal- 
dehyde gas in the laboratory may prevent indigo formation. 

SKATOL. 

Skatol Potassium Sulphate or Skatol. — A certain 
amount of skatol sulphate is found in the urine in conjunction 
with the similar indoxyl compound, indican. This substance will 
produce a red discoloration of the chloroform with the Jaffe test 
which is not removed by a 10 per cent, solution of sodium thiosul- 
phate, as is the pinkish discoloration due to iodin and iodids. 
This substance is also known as indigo red, skatoxyl red, and 
urorubin. It has the same significance as indican. 

EosENBACH^s Test. — Boil a few cubic centimeters of urine, 
and then add drop by drop nitric acid and continue boiling. In 
the presence of skatol a deep-red color appears and the foam 
produced by shaking is a bluish red. 

Amylic Alcohol Test. — Proceed as in the Jaffe test, 
finally overlaying the whole with pure amylic alcohol. Gentle 
agitation will extract the skatol, which will produce a dirty 
brown or black discoloration of the amylic alcohol as it floats 
on the top of the tube. 

Urorosein (Indol-acetic acid) Test. — Technic: Mix about 
5 cubic centimeters of urine with equal parts of C. P. HCl and 
add a few drops of a freshly prepared solution of sodimn nitrite. 
A characteristic rose-red color develops. This pigment also 
gives a red color reaction with Ehrlich's dimethyl-amide ben- 
zaldehyde reagent. 



292 THE URINE. 

THE CHLORIDS. 

The quantity of chlorids in the urine is usually decreased 
in: 1. Most febrile diseases. 2. Nephritis. 3. Wasting dis- 
eases. 4. Pneumonia. 

Approximate Estimation of the Chlorids. — To a few cubic 
centimeters of urine in a test-tube, add a few drops of nitric 
acid, boil and filter to remove the albumin. Add to the filtrate 
a few drops of a 10 per cent, solution of silver nitrate. The 
abundance of the white cloud will roughly indicate the amount 
of chlorids present. 

Accurate Methods of Quantitative Determination. — Method 
of Salkowski-Volhard. Take 10 cubic centimeters of urine in 
a beaker and dilute with 50 cubic centimeters of distilled water, 
and then treat with 4 cubic centimeters of concentrated nitric 
acid and 15 cubic centimeters of a standard silver nitrate solu- 
tion. The mixture is then further diluted with distilled water 
up to 100 cubic centimeters, and after thorough agitation is 
passed through a dry filter. In a carefully measured portion of 
the filtrate the excess of silver is carefully titrated with a solu- 
tion of potassium sulphoc3^anid of such strength that 25 
cubic centimeters correspond to 10 cubic centimeters of the 
standard silver solution. A few drops of a saturated solution 
of ammonio-ferric alum serve as an indicator. The amount of 
silver solution employed to precipitate the chlorids in the 10 
cubic centimeters of urine is then calculated. The number of 
cubic centimeters necessary for this precipitation is multiplied 
by 0.01, which will indicate the amount of chlorids in 10 cubic 
centimeters of the urine calculated as sodium chlorid. The 
presence of albumins or of sugar does not interfere with this 
reaction. (For test solutions see Appendix.) 

PURDY'S CENTRIFUGAL METHOD OF 
ESTIMATING CHLORIDS. 

This method, while having nothing in common with the 
accuracy of the preceding one, is very convenient and has the 
advantage of yielding quick clinical results. 

Ten cubic centimeters of clear, filtered, albumin-free urine 
are placed in a centrifuge tube, which is graduated to 15 cubic 



ORGANIC CONSTITUENTS OF URINE. 293 

centimeters. One cubic centimeter of strong nitric acid and 
4 cubic centimeters of a 5 per cent, solution of silver nitrate 
are then added. The tube is shaken by inversion and the mixture 
allowed to stand for a few minutes, after which it is placed in a 
centrifuge and whirled for three minutes at the rate of 1200 
revolutions per minute. The bulk percentage of silver chlorid 
is then read off;, from which the percentage by weight, both of 
sodium chlorid and of chlorin, equivalent to the precipitated 
silver chlorid may be calculated. One per cent, by bulk repre- 
sents 0.13 per cent, by weight of NaCl and 0.08 per cent, of 
chlorin. 

As previously stated, the amount of chlorin in the urine 
depends upon the amount ingested, ranging normally between 10 
and 15 grams in twenty-four hours. 

THE ORGANIC CONSTITUENTS OF THE URINE. 

GENERAL CONSIDERATIONS. 

The normal organic constituents of the urine comprise the 
normal end-products of nitrogenous metabolism within the body, 
also various products of albuminous putrefaction which have 
found their way into the general circulation from the intestinal 
tract. Finally constant pigments which bear a relation to the 
normal blood-pigments and various other substances of obscure 
origin. 

Under abnormal conditions there may appear other normal 
constituents of the blood which do not ordinarily find their way 
into the urine, while in pathologic conditions various products 
of abnormal metabolism appear. 

Just as the composition of the average dietary varies in 
different localities, and is modified from time to time by design 
or by seasons, so wilVthe urine show material variations in its 
normal end-products of nitrogenous metabolism. Much clinical 
work has been done in an effort to determine the significance 
of many of these substances, but up to now little of definite 
value is known of most of them; so only those findings which 
are of actual clinical value will be considered on the pages 
following. 



294 THE URINE. 



UREA. 



Urea ((NH2)2C0) was first synthetically prepared from 
ammonium cyanate in 1828 by Woliler. Formerly it was sup- 
posed that urea resulted from uric acid through a process of 
oxidation^ and that this was its only source. Now this error is 
well known, and while it is recognized that the formation of 
urea from uric acid is possible and that a small portion of the 
total amount may be derived in this manner, modern research 
has shown that in man urea is largely derived from the destruc- 
tion of the nucleins within the body, and that the sources of 
the nucleins are both the tissue cells and the cells of animal 
foods ingested. 

Urea is therefore the principal end-product of protein 
metabolism. It is most abundant in the urine of man and of the 
earnivora. It appears in varying quantities in nearly all the 
fluids and many of the tissues. The normal adult male excretes 
in twenty-four hours from 30 to 35 grams. 

It has been repeatedly shown that during the decomposi- 
tion of albumins by means of acids and alkalies, as during the 
process of tryptic digestion and albuminous decomposition, a 
large amount of mono-amido-acids result. And it is supposed 
that these bodies probably represent the intermediary products in 
the transformation of albuminous nitrogen into urea. Under 
certain pathologic conditions these acids may appear in the 
urine, and when they are there noted the elimination of urea is 
much diminished. In health, however, this does not occur, and 
on examination of the different tissues of the body such acids 
are found only in traces. We must conclude, therefore, that 
these acids, supposing them to occur as the primary products 
of albuminous decomposition within the bod}^, are transformed 
at once into other substances, which in turn give rise to urea. 

It has also been shown that the amido acids yield carbamic 
acid on oxidation, and that on alternate oxidation and reduction 
urea can be produced from the ammonium salt (ammonium 
carbamide) . While it is generally assumed that urea is largely 
referable to a transformation of the mono-amido-acids into 
ammonium carbamate, and while it has also been shown that 
such a transformation does actually occur, it must be remem- 



UREA. 295 

bered that at best only traces of these amido acids are found in 
the tissues. 

From these conclusions it is reasonable to believe that the 
greater portion of albuminous nitrogen is set free from the vari- 
ous organs of the body in the form of an ammonium salt of para- 
lactic acid, and that this salt is now generally conceded to be 
the antecedent of urea. 

It is probable that a certain amount of urea is produced 
in the body in a number of ways, and there is ground for the 
belief that its formation is not confined to a single organ. 
The greater part is, without doubt, produced in the liver. In 
corroboration of this fact it has 'been repeatedly shown that in 
disease of this organ, when accompanied by extensive destruc- 
tion of the glandular elements, a diminished amount of urea is 
found in the urine, while ammonia and lactic acid are found 
in increased amounts. In certain cases of this class as much 
as 37 per cent, of the total urinary nitrogen has been found in 
the urine in the form of ammonia. 

If we accept the modern doctrine that urea originates not 
only in different ways, but that it may also be formed in other 
organs beside the liver, then we can understand why it is that 
in certain diseases of the liver the diminution in the amount of 
urea excretion is not always proportionate to the extent of 
parenchymatous degeneration, and that no case has yet been 
reported in which the excretion of urea has ceased altogether. 

Nitrogenous Equilibrium. — The albumins are the ultimate 
source of the urea, and according to Pettenkofer they exist within 
the body in two forms, viz. : as organized albumin, which is built 
up into tissues, and the so-called circulating albumin^ which is 
present in excess of the actual requirements of the body and 
which may be broken down directly and eliminated in the urine 
without ever having entered into the construction of the body- 
proper. This latter portion of the body albumin is said to fur- 
nish the bulk of the urea, while the fixed tissue albumin repre- 
sents the minor but more uniform source. The actual amount 
eliminated will, therefore, be primarily dependent upon the 
quantity ingested. 

Experiment has shown that under normal conditions of 
average diet the total urinary nitrogen is practically equivalent 



296 THE URINE. 

to the quantity ingested, barring a small fraction, which escapes 
digestion and appears in the feces. Such a condition is spoken 
of as the nitrogenous equilibrium of the body. Of this relation 
infinite variations exist, which may even vary from time to time 
in the same individual. If the amount of nitrogenous food is 
suddenly diminished the amount of urinary nitrogen will also 
decrease; then if the reduced ingestion remains constant the 
nitrogen output, while lowered, will at the same time tend to 
remain level. If, on the other hand, the nitrogen intake is in- 
creased, an increased nitrogen elimination speedily follows, 
but here a certain fraction will be retained in the body and 
gradually a higher level of equilibrium will be established. 

There are natural limits to this power of accommodation 
of the system, to nitrogenous ingestion and elimination, so that 
we may find a point which varies in different individuals where 
a further nitrogen ingestion does not lead to a higher level of 
nitrogenous equilibrium, and when, consequently, a further re- 
tention of nitrogen does, not occur. Overfeeding then results in 
various digestive disturbances. Diarrhea and vomiting may 
occur through nature's effort to protect the body from a further 
increase in circulating nitrogen. 

Underfeeding, on the other hand, generally leads to in- 
creased destruction of the organized albumins. Although for a 
while the body's store of fats and carbonates is capable of 
protecting the body against an undue loss of nitrogen in this 
direction, still if the reduced intake of nitrogen continues suf- 
ficiently long, death finally ensues. 

From the fact that the level of nitrogenous equilibrium 
varies in different individuals and in the same individual from 
time to time, it follows that the amount of urea excreted must 
also vary according to the same irregular manner. Any figures, 
therefore, which are supposed to indicate the amount of urea 
eliminated, can be of little and uncertain value unless the actual 
state of the individual's health is known, also his body-weight, 
habits as to exercise, the amount of nitrogenous food ingested, 
etc. Only when we are in possession of an accurate knowledge 
of these several factors can we say whether the urea excretion is 
or is not normal. 



UREA. 297 

Certain figures have been compiled by physiologists to indi- 
cate the amount of nitrogen which should enter into the com- 
position of the diet and from which we may approximately 
calculate the amount of urea that should be excreted. By esti- 
mating this, or still better, by determining the total nitrogen 
elimination, we can then determine whether or not the indi- 
vidual is consuming the proper amount of nitrogenous food in 
his dietary. While such figures may serve as a general guide, 
they have mostly been constructed without due regard for the 
factors above indicated, and should not, therefore, be too im- 
plicitly relied upon. 

According to Simon, among the well-to-do classes the elimi- 
nation of from twenty to t\\^enty-five grams of nitrogen m 
twenty-four hours is about normal, taking the average body- 
weight of the individual into consideration. A smaller amount 
is not infrequently met in persons of sedentary habits who may 
appear to be in perfect health. 

Urea in Disease. — While extensive variations occur in the 
urea excretion of health, still greater variations from the average 
standard are noted in disease. But here also should be taken 
into account the amount of nitrogen ingested in relation to the 
body-weight. 

An increased elimination of urea referable to the destruc- 
tion of organized albumins is frequently observed, but this 
may in cases be obscured by a deficient nitrogen] ingestion un- 
less the total intake of the latter is not definitely known. It 
is of interest here/ to note that in certain diseases of the liver 
in which there is great destruction in the parenchyma, the 
amount of urea may be markedly diminished, even when a fairly 
abundanlj supply of nitrogen is taken in. This will be found 
due largely to the interference in urea synthesis, and as a sec- 
ondary result we find that in these cases a considerable portion 
of the nitrogen appears} in the urine in the form of ammonium 
salts of paralactic acid (see page 294) and of carbonic acid; in 
extreme cases as mono-amido-acids and as leucin and tyrosin. 

Properties of Urea. — ^Urea crystallizes in colorless, quadri- 
lateral, or six-sided prisms with oblique ends, or when rapidly 
crystallized in delicate white needles which melt at 132° C, but 
which are probably decomposed at a temperature of 100° C. 



298 THE URINE. 

They contain no water of crystallization, and are permanent in 
the air, and easily soluble in cold water, in which they form a 
neutral solution. With nitric acid, urea unites to form urea 
nitrate, which crystallizes out in octahedral lozenge-shaped or 
hexagonal platelets. Urea nitrate is readily soluble in distilled 
water, but is soluble^ less readily in water acidified with nitric 
acid. Its formation is frequently observed when urine is exam- 
ined cold, with nitric acid, for the presence of albumin. On 
heating, the crystals are decomposed without leaving any 
residue. 

Detection of Urea. — 1. To detect urea place a drop or two 
of the suspected fluid upon a glass slide, and after adding a drop 
or two of nitric acid, warm gently. If urea is present, after 
partial evaporation and cooling, the microscope will show the 
characteristic crystals of urea nitrate. These are either rhombic 
or hexagonal plates, frequently overlapping like shingles on a 
roof. Their acute angles measure 82°. 

2. Add to the suspected fluid, in a test-tube, a few drops 
of fresh sodium hypobromite. A rapid evolution of gas will 
indicate the presence of urea. 

Since clinical observations are concerned in the total out- 
put of urea, it becomes necessary to estimate the quantity or 
percentage from a sample of twenty-four hours' urine. 

Under normal conditions of average health the percentage 
of urea is two. 

The average daily excretion of urea is 40 grams or 500 
grains, or about half the weight of the total solids. 

Quantitative Estimation of Urea. — ^The quantitative esti- 
mation may be determined by calculation from the observed 
volume of nitrogen gas evolved from a measured quantity of 
urine by a process of decomposition. For practical purposes 1 
gram of urea is estimated to furnish 37 cubic centimeters of 
nitrogen gas. The decomposition of the urea contained in the 
measured volume of urine is accomplished by means of an alka- 
line solution of sodium , hypobromite (see Appendix for formula 
of Knop's solution) . The test is best conducted in a Doremus- 
Hinds ureometer (see Fig. 58). 

The TEST.-^First pass some urine through the small tube 
to wet the stop-cock, then fill the large tube with Knop's solu- 



ESTIMATION OF UREA IN UKINE. 299 

tion^ and the smaller side tube with urine exactly up to the 
cubic centimeter mark. Now, drop by drop, allow exactly 1 
cubic centimeter of urine 'to pass into the reagent in the large 
tube. When the bubbles of gas (nitrogen) cease to rise, the 
fraction of a gram of urea may be read directly from the grad- 
uated scale on the larger tube. 

Example. — Suppose that after admitting exactly 1 cubic 
centimeter of urine the level of the fluid stands at the 0.018 
mark. Then the 1 cubic centimeter of the sample contained 
0.018 gram of urea. To obtain the total amount of urea ex- 
creted in twenty-four hours, it is only necessary to multiply 
this figure by the number of cubic centimeters in twenty-four 
hours^ urine. 

ESTIMATION OF UREA IN THE URINE. 

Marshall's Method.^ — Marshall in 1913, as a result of 
work done in the laboratory of physiologic chemistry at Johns 
Hopkins University, introduced a method of determining urea 
in the urine and other body fluids, depending upon the well 
known fact that urea may be converted into ammonium car- 
bonate by enzyme action. Such enzymes are found in certain 
bacteria, fungi and plants. In the method of Marshall, the 
enzyme of the soy-bean (glycine hispida) discovered by 
Takeuchi^ ini 1910, is used. This method is both accurate and 
simple, requiring very little time, no complicated apparatus 
while it compares favorably in accuracy with the older and 
more cumbersome methods. 

Preparation of Enzyme Solution. — An aqueous solution of 
the soy-bean is prepared by macerating and grinding the bean, 
extracting it with acidulated water (K/10 HCl) and filtering 
until a perfectly clear solution is obtained. This aqueous solu- 
tion may be kept without deterioration for some time, but not 
indefinitely. Cullen and Van Slyke have produced a permanent 
preparation of the ferment by extracting first with acidulated 
water, then precipitating with t^^dce the volume of acetone and 
filtering off the precipitate. This when thoroughly dry pre- 
serves its enzyme activity indefinitely. Such solutions should be 

5 Marshall : Jour. Biol. Chem., 1913, xiv, 3. 

6 Takeuchi : Chem. Zeitschr., 1911, xxxv, 408. 



300 THE URINE. 

alkaline to methyl-orange and any preparation (unless Just pre- 
viously prepared from the powdered bean or the dry extract) 
found not to be distinctly alkaline should have less acid used in 
its preparation, as the activity of the enzyme will be greatly 
reduced in the presence of even faint acidity. At the present 
time it is not even necessary for the clinician to prepare the 
extract himself, as it may be obtained in convenient tablet form 
in the open market.''' 

Principle. — A solution in which the urea is to be deter- 
mined is treated with a solution of the enzyme, and after the 
conversion into ammonium carbonate is complete, the alkalinity 
of the solution is determined by titration, with a standard acid 
solution, using an indicator not sensitive to CO2. From the 
amount of standard acid used, the amount of urea originally 
present in the solution is readily calculated. The practical re- 
sult obtained with this method being within 2 per cent, of the 
absolute. The presence of glucose or protein in urine does not 
interfere with the accuracy of the test. 

Technic. — Two 5-cubic centimeter portions of urine are 
measured into each of 2 flasks of 200-cubic centimeter capacity 
and diluted with 100 to 125 cubic centimeters of distilled water. 
Two cubic centimeters of enzyme solution or one 25-milligram 
urease tablet previously rubbed up in a mortar with 5 cubic cen- 
timeters of ammonia-free water are added to one flask and a few 
drops of toluene to each. If time is no consideration both are 
allowed to remain at room temperature with occasional shaking 
for eight hours, or over night. If time is a consideration two 
tablets may be used and the specimen digested at 40° C. for 
one hour. A most rapid but less accurate determination may 
be made by using 1 cubic centimeter of urine to 100 cubic 
centimeters of distilled water and two 25-milligram tablets. 
Digest at 40° C. for fifteen minutes only before proceeding with 
titration. The fluid in each flask is then titrated to a distinct 
pink color with N/10 HCl using methyl-orange as an indicator. 

Calculation. — ^The amount of HCl required for the con- 
tents of the flask containing the urine and enzyme solution less 
the amount used for 5 cubic centimeters of urine alone, and 



7 Hynson, Westcott, and Dunning : Baltimore, Md. and Arlington Chemical 
Co., Yonkers, N. Y. 



ESTIMATION OF UREA IN URINE. 301 

that previously determined for 2 cubic centimeters of the 
enzyme solution corresponds to the urea originally present in 
the sample of urine. Because 1.0 cubic centimeter of N/10 
HCl is equivalent to 3.0 milligrams of urea, thus the number 
of cubic centimeters required multiplied by 0.6 gives the value 
of urea expressed in terms of grams per 1000 cubic centimeters 
of urine. 

Method of Van Slyke and Cnllen.s — Measure 45.0 cubic 
centimeters of ammonia-free water and add exactly 5.0 cubic 
centimeters of a 24-hours urine specimen. Stop the tube with 
the finger and thoroughly mix. Eemove 5.0 cubic centimeters 
of the diluted urine by means of an accurate pipette and place 
in a large test-tube (see Fig. 27) add 1 or 2 drops of amylic 
alcohol to prevent foaming and 1.0 cubic centimeter of enzyme 
solution or two 25-milligram tablets. Close the tube and allow 
it to stand for fifteen minutes for the enzymei to act. Measure 
into a second tube 25.0 cubic centimeters of N/50 normal solu- 
tion of hydrochloric acid, add 1 or 2 drops of amylic alcohol 
and 1 drop of indicator solution (alizarin or metyl red). Con- 
nect tube A and B as shown in the figure, placing cotton in the 
filter tubes in order to prevent loss of fiuid from one tube to the 
other. Tube A, should be connected with a wash-bottle contain- 
ing sulphuric acid in order to prevent the entrance of ammonia 
from the air of the room. Tube B, containing the standard acid 
solution, is connected with a suitable suction apparatus and for 
about one-half minute aspirated to remove any free ammonia 
present in tube A, then open tube A and add 5.0 cubic; centim- 
eters of a saturated solution of potassium carbonate; imme- 
diately replace the stopper and aspirate until all the ammonia 
haa been carried over into the tube B^ the time required to ac- 
complish this varies with the power of the suction apparatus, 
which should be determined by previous trial for the particular 
apparatus used. After completion of this process the excess of 
acid contained in tube B is titrated against a N/50 sodium 
hydrate solution. 

Calculations. — ^The number of cubic centimeters of N/50 
acid neutralized multiplied by the factor 0.056 will give tlie 



8 Van Slyke and Cullen : Jour. A. M. A., 62-1558, 1914. 



302 THE URINE. 

niiinber of graons of urea plus the ammonia nitrogen in 100 cubic 
centimeters of nrine. 

If desired to determine the urea nitrogen alone, the am- 
monia nitrogen in tlie same sample mnst be determined and 
subtracted from the total as determined above. This may be 
done by the same teohnic except that 5.0 cubic centimeters of 
undiluted urine and no urease are added, finally using the fac- 
tor 0.0056 to multiply the number of cubic centimeters of lSr/50 
acid neutralized. The ammonia tubes may be run in the same 
series as those for urea determination, using the same current 
of air for all. 

URIC ACID. 

General Considerations. — Uric acid, like urea, is nitrogen- 
ous. The normal proportion of uric acid to urea is as 1 : 45. In 
health it exists in solution as sodium and as potassium urate. A 
healthy adult excretes 0.3 to 1.0 gTam of uric acid in the course 
of twenty-four hours. The amount increases physiologically 
with increased ingestion of food, and pathologically with in- 
creased nitrogen metabolism in about the same proportions as 
urea. The amount of uric acid in urine varies directly with the 
specific gravity, so that the last two figures of the specific gravity 
(calculated to four places), multiplied by two, give approxi- 
mately the number of centigrams of uric acid in the litre. The 
daily excretion of uric acid is increased in fevers and in leu- 
kemia. The relation of the elimination of uric acid to attacks 
of gout and the so-called uric acid diatliesis, is still an unsettled 
question. Of necessity the elimination of uric acid is increased 
by the ingestion of uric acid and other pimn bodies, as well as 
by foods rich in nuclein (rich in cells). 

Properties of Uric Acid. — TTric acid is practically insoluble 
in cold water, requiring 18,000 parts of water to dissolve 1 part 
of uric acid. It is freely soluble in alkalies and in solutions of 
the carbonates. Pure uric acid crystals are colorless, transparent 
platelets, the angles of which are frequently irregular and 
rounded off. Such crystals are occasionally seen in freshly 
voided urines, but more commonly they are found in the form 
of brownish-yeUow, whetstone-shaped crystals, occurring singly 
or in gTOups (see Plate IX, a and l) . ITric acid once de- 



URIC ACID. 



303 



posited remains undissolved in acid nrine. Owing to the 
inclusion of the urinary pigments in the crystals, these, when 
present in appreciable quantity, are spoken of as ^^rick-dust/' 

Microscopic Appearance. — The reddish specks observed by 
the naked eye are found, upon microscopic examination, to 
be various modifications of rhombic prisms (see Plate IX, c, d, 
e and /) . The simpler forms have some resemblance to the con- 
ventional lozenge or whetstone. 

Si^ificance. — If these crystals deposit in appreciable quan- 
tities soon after micturition, it 
may be considered as a sign of 
impending gouti or gravel for- 
mation. Such a deposit, however, 
is not necessarily evidence that ^ " \ \ 

the elimination of uric acid is 




E-odl 


-0 


E002 

1003 


r 


-1 

2 




^A^ 


i 


/ 


m 


) 



Pig. 58.— Ureometer. 



excessive. 

Isolation of TJric Acid. — To 

200 cubic centimeters of urine add 
10 cubic centimeters of HCl, and 
let stand for twenty-four hours; 
the uric acid will then have set- 
tled to the bottom of the con- 
tainer, from which it may be 
collected by decanting, filtering, 
and finally washing in cold water. 

Qualitative Tests for TJric Acid. — Mueexid Test : Put the 
solution supposed to contain uric acid or urates in a porcelain 
dish, add a drop of nitric acid and evaporate to dryness. After 
cooling, allow a drop or two of ammonia water to come in con- 
tact with the residue. The presence of uric acid or urates will 
be shown by bright blue or violet (murexid) color. 

ScHiFP^s Test. — ^Having a residue prepared as above, or 
crystals supposed to be uric acid, dissolve in a test-tube with 
the aid of a solution of sodium carbonate. Moisten some filter- 
paper with a 10-per-cent. solution of silver nitrate. Into the 
center of this paper allow a few drops of the uric acid sodium 
carbonate solution to fall. In the presence of uric acid the 
silver nitrate will be reduced to black metallic silver. 



304 THE URINE. 

Approximate Quantitative Determination. — According to 
Gubler, the amount of uric acid in urine may be approximately 
determined by stratifying the specimen of urine to be tested 
upon a layer of nitric acid contained in a test-tube, so that the 
volume of urine to the volume of nitric acid is as 3 : 2. After 
a short interval uric acid crystals will separate out as a cloudy 
white ring at the line of junction of the two fluids. If the 
amount of uric acid in the specimen is increased, the precipita- 
tion will be plain in five minutes or less. If diminished it will 
not appear until later. This determination is of value only 
when the daily excretion of urine is approximately normal, and 
if it is diminished in amount it should be diluted with water 
up to the average amount before applying the test. Obviously 
the conclusions derived from such a method should be given 
very slight weight clinically, and then only when the estima- 
tion is made with a part of the twenty-four hours' collection, 
properly diluted as mentioned above. Albumin must first be 
removed by slight acidulation with dilute acetic acid, boiling 
and filtering, after which the test should be applied when the 
urine has cooled to room-temperature. 

QUANTITATIVE CLINICAL DETERMINATION 
OF URIC ACID. 

Ruhemann's Test.^ — This method is a very convenient 
clinical one, , although its results are by no means as accurate as 
other more complicated methods. What the general practitioner 
desires, as a rule, is to know whether the uric acid is increased 
or diminished, not the absolute quantitative values. This 
method determines the total purin content and is sufficiently 
accurate for clinical purposes. 

It consists in the use of a specially graduated tube, the 
uricometer (see Fig. 59), in which are placed the reagents and 
the urine to be tested. The calibrations etched on the tube are 
devised to give directly the amount of uric acid in parts per 
1000. The method depends on the decolorization of an iodin 
solution by the uric acid of the urine, and the measurement of 
the amount of urine which must be added to a definite amount 
of iodin solution to effect this decolorization. 



9 Webster's "Diagnostic Methods," 1912. 



CLINICAL DETERMINATION OF URIC ACID. 



305 



Teohnio. — Sufficient carbon disulphid is 
placed in the tube to bring the lower meniscus of 
this reagent to the S mark. A solution of iodin in 
potassium iodid is then added, so that the upper 
portion of the meniscus coincides with the mark J. 
(For solution see Appendix, page 499.) 

The urine is added slowly by means of a 
pipette until the lowest calibration is reached. 
The glass stopper is inserted and the contents of 
the tube mixed by repeated inversion for about 
fifteen seconds. The carbon disulphid absorbs the 
iodin, taking on a distinct purple coloration. If 
this amount of urine does not completely decolorize 
the iodin, shown by porcelain-like color of the car- 
bon disulphid solution, more urine is added and 
the tube again inverted for fifteen seconds. This 
process is continued until repeated shaking of the 
tube causes the carbon disulphid to assume a pale- 
pink color. This is the end-point of the reaction, 
as more shaking of the contents will cause the in- 
dicator to assume the characteristic porcelain- 
white appearance. The time consumed by this 
test is from five to sixteen minutes. The amount 
of uric acid is then read off directly from the tube 
in parts per liter. 

Cautions. — Should the urine contain less 
uric acid than can be read off from the calibra- 
tions, a second test is made, adding the iodin 
solution to the mark midway between S and J, 
the amount indicated on the tube being, of course, 
divided by 2. Conversely, should the urine con- 
tain m.ore uric acid than is represented by the 
lower calibration, one adds the iodin solution to 
the point above J and multiplies his reading by 
1.5 or adds the iodin solution to the second mark 
above J and multiplies the reading by 2. 

With this method the urine must be acid in 
reaction. If the urine contains a sediment of 
urates, it should be thoroughly shaken before be- 

20 



Fig. 59.— 
ruhemann's 
Uricometer. 



306 THE URINE. 

ing added, so that the urates may be in suspension. Any free 
uric acid which may have separated in the sediment is not de- 
termined in this method. Strongly colored urines have no 
influence upon the decolorization. The presence of sugar does 
not interfere with the results, but if albumin be present in large 
amounts it should be removed by acidifying with dilute acetic 
acid, boiling, and filtering. 

Method of Folin and Shaffer. — Place 300.0 cubic centim- 
eters of specimen in a large Erlenmeyer flask or beaker and add 
75.0 cubic centimeters of uranium acetate solution (see Appen- 
dix, page 497), stir the mixture and allow to stand for a few 
minutes; this precipitates the phosphates and other substances. 
Filter through double filter paper, and of the filtrate place 125.0 
cubic centimeters in each of two flasks (125.0 cubic centimeters 
of filtrate represents 100.0 cubic centimeters of specimen) and 
add to each, 5.0 cubic centimeters of ammonium hydrate to con- 
vert the uric acid to ammonium urate. Mix well and stand 
aside for twenty-four hours to allow the precipitate to settle to 
the bottom of the beaker, then pour off the clear supernatant 
fluid and collect the precipitate, which is repeatedly washed with 
the ammonium sulphate solution until the wash-water is free 
of chlorin, as shown by the addition of 10 per cent, silver nitrate. 
Puncture the filter paper and wash precipitate into a beaker with 
approximately 100.0 cubic centimeters of distilled water. Add 
15.0 cubic centimeters of concentrated sulphuric acid, stir con- 
stantly and titrate the mixture immediately with the previously 
prepared permanganate of potash solution (see Appendix, page 
497), until a faint pink color appears and remains for a few 
seconds. 

Calculation. — Each cubic centimeter of N/20 perman- 
ganate solution is equivalent to 3.75 milligrams of uric acid. 
This multiplied by the number of cubic centimeters of perman- 
ganate used gives the uric acid in 100.0 cubic centimeters of 
urine from which the twenty-four hours' total may be calculated. 
Correction: Ammonium urate is slightly soluble in water 
therefore, a correction of 3 milligrams should be made for each 
100.0 cubic centimeters of urine. 



THE URATES. 307 

THE PURIN BASES IN THE URINE. 

These comprise xanthin, hypoxanthin, heteroxanthin, para- 
xanthin, guanin, and adenin. These bases are normally present 
in the urine and comprise approximately one-tenth as much as 
the normal uric acid. The amount of purin bases normally 
found in the urine in twenty-four hours varies between 0.028 
and 0.058 gram. There is at present no practical clinical 
method of estimating them that is within the scope of the 
average laboratory equipment. 

THE URATES. 

Greneral Consideration. — When the urine cools the urates 
may settle to the bottom of the container as a cloudy reddish- 
yellow precipitate. This is most likely to occur when the urine 
is scanty, concentrated, and highly acid. This condition often 
obtains in fevers, and in congestion or inflammation of the kid- 
neys. The sediment then presents a fairly characteristic appear- 
ance, being clay-colored, reddish yellow, or rose-red. It may 
adhere to the walls of the vessel as a fine, reddish coating. 

The ordinary uratic sediment consists of a mixture of the 
urates of sodium^ potassium, calcium, magnesium, and ammo- 
nium. Sodium urate predominates. With the exception of 
ammonium urate, these urates only appear in acid urine. 
Uratic sediments often contain a few crystals of uric acid 
which have been formed during the double rearrangement of 
molecules which resulted in the precipitation of the urates. If 
urine containing a uratic sediment is decomposed by ammo- 
niacal fermentation, the sediment will be changed to acid-am- 
monium urate, and this latter is the only urate sediment which 
occurs in alkaline urine. 

If freshly voided urine is kept in a cool place, the precipi- 
tation of urates will occur rapidly. This same precipitation 
occurs at a higher temperature if the amounts of urates is in 
excess of normal or if the urinary acidity is decreased. This 
precipitate is readily distinguished from phosphates by its 
prompt disappearance upon the application of heat. 

Qualitative Tests.— 1. Half fill a test-tube with turbid urine 
and apply heat to the upper part. If the turbidity is due to the 



308 



THE URINE. 



presence of urates, the heated portion of the urine immediately 
becomes clear. 

2. To some urine in a test-tube add some liquor potassii, 
when the turbidity due to urates promptly disappears. 

Microscopic Appearance of Urates. — The uratic deposit is 
composed of fine, somewhat regular granules, usually occurring 
in groups; sometimes the granules show spiny projections. 
(See Fig. 60.) Ammonium urate occurs only in alkaline urine, 
and is generally accompanied by a copious precipitation of triple 




Fig. 60.— Urate of Soda and Crystals op Uric Acid {h), Oxalate 
OF Lime (o), and Cystin (c). x 350. 

phosphates. Ammonium urate appears as opaque brownish-red 
spherules with or without projecting spines. 

Significance. — The excess of urates is of no special im- 
portance ; they are increased in most conditions accompanied by 
fever, and in many disturbances of metabolism. 

HIPPURIC ACID. 

Hippuric acid, in combination with alkaline bases, is a nor- 
mal constituent of the urine. The average quantity eliminated 
in twenty-four hours is one gram. This amount may be in- 
creased by exercise, by a vegetable diet, and by ingestion of ben- 
zoic acid. In cases where the total excretion of urine is greatly 



PLATE VIII. 






^■? JS ' -ii^-s-^r :-- J: 'S^' ^^" '"^' ^ 



f : 



u.*. 






(yl& 



^%. 



{■A !-r\ 



i.i 



J^ 



/^ 



^ 









Uric Acid Crystals with Amorphous Urates. X 450. 
(After Peyer. ) 



CREATININ. 309 

diminished, hippnric will be spontaneously thrown out of solu- 
tion. This is, however, a rare occurrence, because hippuric acid 
is readily soluble in water. 

Microscopic Appearance. — The crystals are characteristic 
rhombic prisms, resembling in a measure the coffin-lid crystals 
of triple phosphates. Hippuric acid crystals may, however, be 
distinguished by the fact that they are precipitated in acid 
urine only, and also because they do not dissolve on the addi- 
tion of acetic acid. Phosphates, on the other hand, are precipi- 
tated only in neutral or alkaline urine, and are readily soluble 
in dilute acetic acid. 

CREATININ. 

This normal urinary constituent is present in the twenty- 
four hours' specimen to the amount of one gram. Creatinin is 
derived from the creatin of muscle. It is distinguished in the 
urine by its union with mercuric chlorid, with which salt it 
forms insoluble compounds. Similar characteristic compounds 
are formed with zinc chlorid and silver nitrate. The zinc chlo- 
rid combination has a characteristic appearance, by which this 
substance may be recognized. Under the microscope the zinc 
chlorid combination of creatin appears as minute needles 
arranged in balls and rosettes. Creatinin reduces alkaline copper 
solutions, and therefore affects, in a slight degree, the accuracy 
of the quantitative sugar estimations which depend upon the 
reducing power of sugar-containing urine. 

Weyl's Test. — Add a few drops of a very dilute aqueous 
solution of freshly dissolved sodium nitroprussid and a few drops 
of dilute sodium hydrate solution to 8 to 10 cubic centimeters of 
urine in a test-tube. In the presence of creatinin a ruby-red 
color appears, which changes after a short time to an intense 
yellow. If this solution be heated with a little glacial acetic 
acid, the yellow will change to green and finally blue. Acetone 
gives a similar reaction, but on the addition of acetic acid 
changes to a purplish red instead of green. If the urine be 
heated previous to the application of this test, the acetone will 
be driven off. This test is sensitive to about 1 part in 1700. 

Jaffe's Test.— To the urine to be tested, add a few drops of 
a saturated watery solution of picric acid and a few drops of 10 



310 THE URINE. 

per cent, sodium hydrate solution. If creatinin be present a 
red color appears immediately, which increases in intensity and 
remains permanent for a long time. If glacial acetic acid be 
added the color becomes yellow. Acetone gives a reddish-3^ellow 
color of less intensity than that produced by creatinin. Glucose, 
if present, may give a red color, especially if the mixture be 
warmed. This test is positive for 1 part of creatinin in 5000. 

OXALIC ACID. 

Oxalic acid is normally present in the urine only in very 
small amounts and it appears in combination with calcium as cal- 
cium oxalate. The normal amount of this substance in a urine of 
normal volume is held in solution by the acid sodium phosphate. 
Since oxalic acid requires 500,000 parts of water to effect solu- 
tion of 1 part calcium oxalate, even the slightest variation in 
the normal relation results in its appearance as a deposit. Arti- 
ficially the crystals may be thrown down by carefully neutral- 
izing the specimen with dilute ammonia water, or by simply 
allowing the urine to stand for a time exposed to the air. The 
sediment of calcium oxalate is usually so scant that it is only 
recognized by the aid of the microscope. The more rapid the 
formation of these crystals in the urine after voiding, the smaller 
will the individual crystals appear; this will, in a way, indicate 
whether or not the presence of the crystals is due to a pathologic 
increase in the oxalic acid content, or merely to a change 
of reaction (loss of acidity) from standing. 

Microscopic Appearance. — Oxalates are recognized in one 
of two forms: perfect octahedra, or in hour-glass and dumb- 
bell forms. (See Plate X, a and h, and Fig. 60.) 

Significance. — These deposits sometimes follow the eating 
of stewed rhubarb or other acid fruits containing oxalates.- 
Usually their existence in freshly voided urine indicates imper- 
fect oxidation of retarded metabolism within the economy. 

QUANTITATIVE DETERMINATION OF 
OXALIC ACID. 

Baldwin^s Method. — Five hundred cubic centimeters of 
the twent3^-four-hour specimen are mixed with 1250 cubic centi- 
meters of 95 per cent, alcohol in order to precipitate the calcium 



ESTIMATION OF TOTAL NITROGEN. 311 

oxalate. This mixture is set aside for forty-eight hours, and 
then filtered, care being taken to remove the crystals from the 
walls of the beaker by a rod the tip of which is protected by a 
rubber tube. The sediment is washed thoroughly in a small 
beaker and treated with a few cubic centimeters of dilute hydro- 
chloric acid. The filter is then washed with hot water until 
there is no further acid reaction, and the washings are collected 
and evaporated to 20 cubic centimeters. A little calcium chlo- 
rid solution is added to insure an excess of calcium. The hydro- 
chloric acid is neutralized with ammonia, and then the solution 
is rendered slightly acid with acetic acid. Strong alcohol is 
added in an amount equal to one-half the volume of the fluid, 
and the whole is set aside for another forty-eight hours. The 
sediment of calcium oxalate is collected on an ash-free filter, 
washed with cold water and with a 1 per cent, acetic acid until 
free from chlorids; the filter is incinerated, cooled, and weighed. 
The ash is calcium oxid, each gram of which represents 1.6 
grams of oxalic acid. 

ESTIMATION OF TOTAL NITROGEN IN URINE. 

Kjeldahl's Method. — Accurate determinations of the 
total nitrogen of the urine is best made by the Kjeldahl method 
employing the modifications of Salkowski. The principle of the 
method is, that the organic elements of the urine are reduced by 
oxidation, when the urine is heated with concentrated H2SO4 
and that the nitrogen of those substances which do not contain 
it combined with oxygen, appears as ammonium sulphate, the 
urea being changed directly into CO2 and ammonia. 

The nitrogen is then estimated by liberating the ammonium 
from the acid solution by adding potassium hydrate, distilling 
it off, and passing it into a measured amount of acid. The acid 
solution is then titrated with a known alkali and the difference 
determined in terms of nitrogen. 

Teclinic. — Ten cubic centimeters of urine (or 5 cubic 
centimeters if of high specific gravity) are put in a long neck 
round bottom (Kjeldahl) flask (Fig. 28), to which is added 10 
cubic centimeters of the sulphuric acid mixture (500 cubic 
centimeters cone, nitric acid and 100 gTams of phosphoric 



312 THE URINE. 

anhydrid), immediately followed by the addition of 0.5 gram of 
powdered mercuric oxid to the contents of the flask. The mer- 
cury acts as a catalyzer and aids in the oxidation. The flask 
is then placed on a wire-gauze over a low Bunsen flame and 
boiled for fifteen to twenty minutes after it has become abso- 
lutely colorless (any yellow tinge indicates incomplete oxida- 
tion) . The fumes which arise during the boiling should be car- 
ried away by a fume chamber, or by means of an inverted fun- 
nel, connected with a suitable suction apparatus. When cool 
transfer the liquid to a distilling flask and add 200 cubic 
centimeters of ammonia-free water, sufficient of a concentrated 
solution of l^aOH to neutralize the H2iS04 and a few frag- 
ments of granulated zinc to prevent bumping, during distilla- 
tion, and a small piece of paraffin to reduce the tendency to 
froth. Connect the flask with a condenser, so arranged to pass 
the distillate through a broad delivery tube into a vessel con- 
taining a measured quantity of E'/IO II2SO4 (volume depend- 
ing on nitrogen content of urine) so that the tube is well be- 
low the surface of the N/IO solution. Mix the contents of the 
distillation flask thoroughly and distill the mixture until the 
column has been reduced about one-half. Titrate the partially 
neutralized N/10 H2SO4 by means of N/10 ISTaOH using 
congo-red as an indicator. 

Determination: Calculate the nitrogen content of the 
twenty-four hours' specimen as follows : — 

One cubic centimeter of N/lO NaOH is equivalent to 
0.0014 gram of nitrogen. Subtract the number of cubic centim- 
eters of N/10 NaOH used in the titration from the number of 
cubic centimeters of N/10 H2SO4 employed. The remainder 
is equivalent to the number of cubic centimeters of N/10 H2SO4 
neutralized by the ammonia of urine. 

Therefore, if Y represents the volume of urine used in the 
determination and Y' the number of cubic centimeters of 
N/10 H2SO4 neutralized by the ammonia of the urine, then the 
following proportion is evolved: — 

Y:100::Y' X 0.0014: X (percentage of nitrogen in 
urine). From this the total quantity of nitrogen in the 
twenty-four hours' urine specimen may be determined. 



ESTIMATION OF TOTAL NITROGEN. 313 

FoLiN- Farmer Method. — This method belongs to the so- 
called microchemical methods, since it is adapted to the deter- 
mination of small amounts of nitrogen (about 1 milligram). 

Diluted urine is decomposed with the sulphuric acid mix- 
ture as in the Kjeldahl method. The ammonia formed is then 
liberated by the addition of an alkali and carried over into an 
acid solution by means of a current of air. The ammonia solu- 
tion is then treated with the Kessler- Winkler reagent (see Ap- 
pendix, page 501) and the color compared with a standard solu- 
tion of an| ammonium salt treated in the same way. 

Technic. — Place 5 cubic centimeters of urine in a 50-cubic 
centimeter volumetric flask. Fill the flask to the 50 cubic 
centimeter mark with distilled water and thoroughly mix. 
Transfer exactly 1 cubic centimeter of the diluted urine to a 
large Jena glass test-tube and add to this 1 cubic centimeter 
concentrated H2SO4, 1 grain of potassium sulphate, a pinch of 
powdered mercuric oxid (or 1 drop of 5 per cent. CUSO4 solu- 
tion) and a glass bead or clean pebble to prevent bumping. 
Boil the mixture for two minutes after the mixture has become 
colorless, allow to cool to a syrupy consistence, but do not allow 
to solidify. Then add an excess of ]SraOH;(3 cubic centimeters 
of a 50 per cent, sol.) and aspirate the liberated ammonia by 
means of a rapid current of air (either suction or force-pump) 
into a 100-cubic centimeter volumetric flask containing 20 cubic 
centimeters of ammonia-free water and 2 cubic centimeters of 
N/10 HCl. This requires from eight to ten minutes. Dilute 
the contents to about 60 cubic centimeters with ammonia-free 
water. At the same time dilutd 1 milligram of nitrogen ini the 
form of ammonium sulphate in a second volumetric flask (see 
Appendix, page 504). Nesslerize the two solutions simulta- 
neously with 5 cubic centimeters of Nessler-Winkler, im- 
mediately before using dilute it with 25 cubic centimeters of 
ammonia-free water to prevent turbidity. Immediately fill the 
two flasks with ammonia-free water to the 100 cubic centim- 
eter mark, mix well and determine the relative intensity of the 
two colors by means of a colorimeter, being careful to remember 
which cylinder of the Duboscq contains the solution of known 
strength. It is advisable to use dift'used daylight. If this is not 
available, light from a blue-bulb nitrogen-lamp may be used. 



314 THE URINE. 

When -using yellow light it should pass through a thin sheet of 
white paper before reaching the reflector of the colorimeter. 
Calculation. — The reading of the standard divided by the 
reading of the unknown gives the reading of nitrogen in milli- 
grams in the volume of urine used. From this the total twenty- 
four hours' nitrogen output may be calculated. 

AMMONIA. 

General Considerations. — This substance, although chemi- 
cally belonging in the class of inorganic compounds, is so closely 
related to the nitrogenous metabolism that it is best discussed 
under this heading. 

Ammonia is one of the most important products of protein 
metabolism. It is constantly present in small amounts in nor- 
mal urine, averaging about 0.85 gram of NH3 in twenty-four 
hours, representing from 4 to 5 per cent, of the total nitrogen. 
It is present in combination with various acids and may repre- 
sent largely a portion of the nitrogen which has not been trans- 
formed into urea, but has been used to combine with acid 
substances formed in the protein metabolism of the body. Any 
increase in the production of acid in the system or any increased 
intake of non-carbonate-forming acids will lead to an increased 
excretion of ammonium salts. This is an important factor in 
the metabolism of conditions associated with acidosis. 

Significance. — The total output of ammonia will vary 
ordinarily with the diet; that is, proportionate to the intake of 
total nitrogen. While the increase of the total nitrogen of the 
urine on increased nitrogen intake is largely in the form of urea, 
yet a small increase in the absolute amount of ammonia must 
occur. Likewise we may observe a diminished intake of nitrogen, 
which, while reducing the absolute amount of ammonia, yet 
increasing it relatively. Thus, Folin finds that with a total 
excretion of 16 grams of nitrogen, an ammonia output of only 
0.85 gram (4.3 per cent.), while on a nitrogen-free diet a total 
nitrogen output of 3.6 grams was observed, with an ammonia 
elimination of 0.51 gram (11.3 per cent.).i^ 

Quantitative Method oe Schlosing. — This method is 
most commonly used, and is here given, as it is fairly simple, 

10 Webster's "Diagnosis." 



AMMONIA. 315 

though open to the objections that it is time consuming and 
does not 3'ield absohitely accurate results. 

Technic. — Twenty-five cubic centimeters of urine are placed 
in the vessel (preferably a Petri dish). Above this is placed 
a glass triangle upon which rests a dish containing 20 cubic 
centimeters of tenth-normal sulphuric acid. Twenty cubic centi- 
meters of milk of lime are then poured into the dish containing 
the urine and the whole covered with a bell-jar, the borders of 
which have been well greased to make an air-tight union when 
the jar is placed upon the glass plate. This apparatus is then 
allowed to stand at room temperature from four to five days, 
during which time the ammonia, liberated by the action of the 
milk of lime upon the ammonia salts of the urine, will be taken 
up by the sulphuric acid in the vessel. At the end of this time 
the bell-jar is removed, the acid titrated with tenth-normal 
sodium hydrate, and the number of cubic centimeters of re- 
maining acid determined. One cubic centimeter of tenth-nor- 
mal sulphuric acid neutralized by the evolved ammonia repre- 
sents 0.001704 gram of ammonia. This figure is multiplied by 
4 to obtain the percentage ammonia value. If any moisture is 
present on the inside of the bell-jar it should be washed into the 
sulphuric acid before titration. ^ 

Formalin Method. — ^This method, originated by Eonchese 
and Malfatti, depends on the fact that a solution of an am- 
monium salt, treated with formaldehyd, decomposes with the 
formation of hexamethylenetetramin, the acid combined with 
the ammonia being liberated. This can then be determiDed by 
titration. 

Dilute 10 cubic centimeters of urine with 50 cubic centi- 
meters of water, add 2 or 3 drops of a 1 per cent, alcoholic solu- 
tion of phenolphthalein, and neutralize with ^° NaOH. Five 
cubic centimeters of formalin, previously neutralized with --- 
NaOH, are added to the neutralized urine and the mixture is 
again titrated with ^ NaOH to the appearance of a faint 
permanent pink color, the amount of alkali used being noted. Tt 
is evident that 1 cubic centimeter of this j° NaOH is equiva- 
lent to 1 cubic centimeter of [^ Nil-, or, in other words, 
11 Ibid. 



316 THE URINE. 

represents 0.001704 gram of NH3. Multiply the number of 
cubic centimeters of f^ NaOH used by this factor to obtain 
the NH3 in 10 cubic centimeters of urine. 

Caution. — It has been found that formalin combines, also, 
with the NH2 group of the amiho-acids, thus leading to results 
higher than those of the Folin method. If the figure obtained 
by the Folin method be subtracted from that of the formalin 
method, the result is the NII2 referable to amino-acids. 



Fig. 61.— Folin's Ammonia Apparatus. 

Folin's Method. — Principle : The ammonia of the urine 
is set free by the addition of an alkali and this ammonia is 
then carried over by an air current into a flask containing a 
measured amount of standard acid. The excess acid is then 
titrated. The necessity for distillation is avoided. 

Procedure. — Place 25 cubic centimeters of urine in an 
aerometer cylinder, 30 to 40 centimeters in height (Fig. 61), 
add about 1 gram of dry sodium carbonate and introduce some 
crude petroleum to prevent foaming. Insert into the neck of 
the cylinder a rubber stopper provided with two perforations, 
into each of which passes a glass tube, one of which reaches 
below the surface of the liquid. 'The shorter tube (10 centim- 
eters in length) is connected with a calcium chlorid tube filled 
with cotton, and this tube is in turn joined to a glass tube 



CYSTIN. 317 

extending to the bottom of a 500-cubic centimeter wide-mouthed 
flask which is intended to absorb the ammonia and for this pur- 
pose should contain 20 cubic centimeters of N/IO sulphuric acid, 
200 cubic centimeters of ammonia-free distilled water and a few 
drops of an indicator (alizarin-red or congo-red). To insure 
the complete absorption of the ammonia the absorption flask 
is provided with a Folin improved absorption tube (Fig. 26), 
which is very' effective in causing the air passing from the 
cylinder to come into intimate contact with the acid in the 
absorption flask. In order to exclude any error due to the pres- 
ence of ammonia in the air a similar absorption apparatus to 
the one just described is attached to the other side of the aero- 
meter cylinder, thus insuring the passage of ammonia-free air 
into the cylinder. With an ordinary filter-pump and good water 
pressure the last trace of ammonia should be removed from the 
cylinder in about one and one-half „hours. The number of cubic 
centimeters of the N/10 sulphuric acid neutralized by the 
ammonia of the urine may be determined by direct titration 
with N/10 sodium hydroxide. 

Calculation. — Subtract the number of cubic centimeters of 
N/10 sodium hydroxid used in the titration from the number of 
cubic centimeters of N/10 sulphuric acid taken. The remainder 
is the number of cubic centimeters of N/10 sulphuric acid neu- 
tralized by the NH3 of the urine. One cubic centimeter of N/10 
sulphuric acid is equivalent to 0.0017 gram of NH3. There- 
fore if Y represents the volume of urine used in the determina- 
tion and Y' the number of cubic centimeters of N/10 sulphuric 
acid neutralized by the NH3 of the urine, we have the following 
proportion : 

Y : 100 : : Y' X 0.0017 : X (percentage of NH3 in the 
urine examined). 

From this one may calculate the quantity of NH3 in the 
twenty-four hour urine specimen. 

CYSTIN. 

This substance, in exceedingly small amounts, is a nor- 
mal constituent of urine. It is almost insoluble in water, 
and in the very rare cases where it is present in increased 



3 IS THE UKINE. 

amount it is deposited. To the naked eye the deposit is abun- 
dant and somewhat resembles that of urates. It is, however, not 
dissolved by heat or by the vegetable acids, but is readily dis- 
solved in ammonia water. The ammoniacal solution exposed 
on a slide and examined under the microscope slowly develops 
crystals in the form of hexagonal plates. (Plate XI, d.) Iodo- 
form, which has found its way into the urine from surgical 
dressings, may be mistaken for cystin. 

Tests. — When urine containing cystin undergoes decompo- 
sition, it develops the odor of hydrogen sulphide. When boiled 
with a solution of lead oxid in sodium hydrate, black-lead sul- 
phid is formed. 

LEUCIN AND TYROSIN. 

These are present in the urine in cases of acute yellow 
atrophy of the liver, typhoid fever, and phosphorus poisoning. 
(Plate XI, e and /.) 

To examine for these substances, the urine must be concen- 
trated on a water-bath, and the cooled liquid examined under 
the microscope. 

Microscopic Appearance. — Leucin appears as greenish-yel- 
low spheres with concentric markings or radiating spines. Tyro- 
sin as feathery sheaves, which resemble the frayed end of a rope. 



FART IL 
ABNORMAL CONSTITUENTS OF THE URINE. 

ALBUMIN. 

General Considerations. — Albumin is probably present in 
minute quantities in all urine^ since urine always contains a 
variable number of cellular elements derived from the urinary 
tract. Thus^ even under normal conditions, by means of suffi- 
ciently delicate tests, we can always demonstrate the presence 
of albumin. This amount is, however, so small that it returns 
a negative result with the tests commonly employed in the clin- 
ical laboratory for the detection of albumin in urine. There- 
fore the presence of demonstrable amounts of albumin, by the 
tests now to be described, must be considered as variations from 
the normal. The "normal'^ albumin is nucleo-alhumin, and its 
clinical significance is slight, even when present in an appre- 
ciable quantity. 

Several other albumins are found in the urine, of which 
serum-aTbumin and serwn-globuliji are of the greatest clinical 
importance, and the term albuminuria is understood to indi- 
cate the presence of one or the other, more often of both, in the 
urine. Other albumins which may be found in the urine, be- 
sides those referred to above, are albumose and fibrin. 

The diagnostic and clinical importance of aJbumosuria have 
not yet been determined. Traces have been found in many of 
the infectious fevers. A large albumosuria may be significant 
of multiple myelomata, since in this affection large amounts are 
frequently observed. It has also been found in osteomalacia 
and in nephritis. 

Fibrin, when present, indicates the direct entrance of 
blood-plasma into the urinary tract. It must be distinguished 
from blood-clots, which come under the head of hematuria. 

Causes of Transient Albuminurias. — Physiologic or Func- 
tional Albuminuria: The presence of easily demonstrable 
amounts of albumin can hardly be considered normal under any 
circumstances. However, the presence of a functional albu- 

(319) 



320 THE URINE. 

minuria is recognized by some authorities. An albuminuria 
does, however, occur, in which the organic change in the kidney, 
if there be any, is so slight and evanescent as to be unimportant. 
This form of albuminuria, compared with that caused by severe 
and permanent kidney change, may, with propriety, be termed 
functional, but never, physiologic. 

FuNCTioxAL. — Albuminuria may occur after violent exer- 
cise, after a cold bath, after severe mental strain, and after over- 
eating of proteid food-stuffs, particularly eggs. 

AYhether or not the disturbance giving rise to the albumin- 
uria is insignificant and transient (renal h3^peremia or anemia), 
or partakes of the gradual and progressive variety due to perma- 
nent alteration in kidney structure, to which the term chronic 
nephritis is applied, must be determined by the history, symp- 
toms, and progress of the case. 

Fevers. — Here the occurrence of albumin in the urine is, 
in all probability, dependent largely upon the intensity of the 
infective process, causing irritation of the kidney epithelium, 
incident to the passage of toxic substances circulating in the 
blood, through the glomeruli and tubules of the kidneys; the 
inflammatory process so induced being indicated by the amount 
and duration of the albuminuria. 

Specific Blood-Changes. — The nature of the changes in 
the blood occurring in scurvy, peliosis, purpura, hemophilia, and 
pernicious anemia, is obscure in relation to the production of 
albuminuria. 

Foreign Substances in the Blood. — This form of albu- 
minuria is seen after the excessive ingestion of lead, mercury, 
ether, chloroform, etc. These probably produce the albuminuria 
by irritation of the kidney through efforts of that organ at 
elimination of the substances themselves, or their oxidation 
products. 

Circulatory Disturbances. — Here the passive congestion 
occurring in cardio-vascular and pulmonary diseases directly 
affects the permeability of the renal epithelium. 

Neurotic. — This term applies to albuminuria occurring 
during or immediately succeeding attacks of apoplexy, migraine, 
tetanus, delirium tremens, and certain head injuries. It is prob- 
ably toxic in origin. 



ALBUMIN. 321 

Extea-Eenal. — The presence of blood, pus, chyle or 
lymph, which has entered the urinary tract in appreciable quan- 
tities, will cause albumin to appear in the urine. Eecently 
attention has been called to a form of albuminuria resulting 
from abnormalities in the function of the genital tract. This 
form comes and goes irregularly, and does not show the pres- 
ence of sperm atozoa.i 

In the majority of the above conditions the quantity of 
albumin is small and varies from day to day. Under these cir- 
cumstances the relative clinical importance of the finding will 
be determined by microscopic examination of the urine, aided 
by the history and course of the disease in which it occurs. 

These small and transient albuminurias are of considerable 
importance from the standpoint of life insurance, since too much 
importance is frequently placed on the finding of a trace of 
albumin, particularly in a single specimen, which is usually 
voided succeeding a full meal. 

Qualitative Tests for Albumin. — In detailing the following 
methods for determining the presence of albumin in the urine, 
no attempt will be made to offer a great variety of tests. The 
principle involved in the majority is the same, and depends on 
the coagulation of albumin in the specimen by heat or by chem- 
ical reagents. 

Before proceeding to make the tests, the urine must be 
filtered or centrifugated to remove the turbidity. If this is due 
to large numbers of bacteria it cannot be entirely removed by 
this process. The persistence of a bacterial turbidity will inter- 
fere with the delicacy of the tests. 

1. Boiling and Acetic Acid. — This is the best and sim- 
plest test for routine laboratory work. If albumin is found it is 
well to corroborate the finding by another method. The urine 
should not be alkaline in reaction. 

Tlie Test. — Fill a narrow test-tube almost to the top with 
clear urine, boil the upper third, being careful to avoid too much 
agitation. In the presence of albumin, or the neutral earthy 
phosphates, or of both, a white cloud will appear in the heated 
area. Now, upon the addition from a pipette of a few drops of 



1 William Glenn Young : New York Med. Jour., Jan. 5, 190'i 

21 



322 THE URINE. 

10 per cent, acetic acid, the cloud, if due to phosphates, will 
vanish, while if caused by albumin it will remain and will per- 
haps be increased in density. The chief advantages of this 
method are its simplicity and delicacy, and the fact that it allows 
a comparison between the treated and untreated urine in the 
same tube. The test is most satisfactory when performed in 
strong daylight with the tube held close to a dull-black back- 
ground. 

Caution. — At times the addition of more acid to a urine 
already acid will convert the albumin into an acid albuminate, 
which is not coagulable by heat. To overcome this the acetic 
acid should always be dilute (10 per cent.), and should never 
be added to acid urine before boiling, and then only in suffi- 
cient quantity to produce the desired reaction. 

2. Heller's Test. — This test is best performed with the 
Meeker albumin tube. 

The Test. — Allow about one-half inch of warm, clear urine 
t0| enter the tube through the small curved top. Place the fore- 
finger over the upper end of the tube to retain the urine, and 
dry with care the outside of the tube; plunge it immediately 
into nitric acid of sufficient depth to allow the acid to flow in 
and elevate the urine above it. If this is done with care the 
line of demarkation between the urine and the acid will be very 
sharp, and the presence of albumin will be shown by a white 
line at the plane of contact. Five minutes should be allowed 
for the appearance of the reaction, the delicacy of which is in- 
creased if both the urine and the acid are previously warmed to 
about 60° C. (a temperature too high to be endured by the hand 
for more than a few seconds). 

A roughly quantitative test by Heller's method is suggested 
by D. W. Prentiss. 2 

Technic. — 1. Make the underlying nitric test for albumin, 
in a test-tube. 

2. Allow the tube to stand two minutes. 

3. Hold the tube between a black or dark object and the 
eyes, on the level with the eyes. 

4. Note the density of the, ring of albumin at the line of 
contact. 



2 Med. Council, Philadelphia, August, 1912. 



ALBUMIN. 323 

Sources of Error. — If a cloud appears at the line of con- 
tact in a urine known to contain an excess of urates, this source 
of error can be eliminated by diluting the urine with one or two 
volumes of water, and repeating the test. 

Nitric acid precipitates certain resins which appear in the 
urine after they have been administered for medicinal purposes. 
If these cannot be excluded another test should be employed. 
If an excess of phosphates exist they may be held in solution 
by the addition of a few drops of a 10 per cent, solution of 
acetic acid. 

Nucleo-albumin may be held in solution by the addition of 
one-sixth volume of saturated solution of NaCl, and then pro- 
ceeding as before. 

Purdy's Test. — Mix a half -inch of Purdy^s reagent (see 
Appendix) with four inches of urine in a six-inch test-tube; if 
a whitish cloud appears either at once or after standing, the 
urine contains albumin. 

Tanret's Test. — Acidify a quantity of urine with dilute 
acetic acid; if mucin produces a cloud, filter and to the filtrate 
add about 5 cubic centimeters of alcohol and heat slightly. 
Now substitute this prepared urine for the urine used in the 
description of the nitric acid contact- test, using Tanrefs 
reagent in place of the nitric acid (see Appendix) ; the pres- 
ence of a white line at the level of contact denotes albumin. 

Ulrich's Test. — The specimen should always be filtered. 
The reagent used is a saturated solution of common salt, acidified 
with 1 per cent, acetic acid. A few cubic centimeters are poured 
into a test-tube and boiled. The urine is then allowed to flow 
over the hot acid brine from a pipette, forming a sharp line of 
contrast. The hot brine will coagulate the albumin, while the 
acid will prevent the precipitation of the phosphates. The 
presence of salts prevents the precipitation of "nucleoalbumin." 

Caution. — ^The phosphates are sometimes precipitated by 
this method, and it is best to add to the urine as a routine 
measure about one-sixth volume of the acid salt solution before 
applying the test. 

Quantitative Estimation of Albumin. — Method of Esbach : 
For clinical purposes as a ready means of comparing the albu- 



324 



THE URINE. 



min content in a given case or a number of cases, this method 
will be found applicable and convenient. 

The amount of gravitated precipitate should never be con- 
founded with the actual percentage of albumin as determined by 
accurate weighing methods, which rarely amounts to more than 
4 or 5 per cent., while by the method of Esbach the moist precip- 
itate may exceed half of the bulk of the fluid in the tube. 

The Test. — Fill the Esbach albuminometer tube (see Fig. 
62) with urine to the line marked ^^U/^ and fill with Esbach 
reagent (for preparation see Appendix) to the 
-^ line marked "E.^^ The tube is then closed with a 

rubber stopper, and the liquid thoroughly mixed by 
repeated inversion without shaking, . and is then 
set aside in a vertical position for twenty-four 
hours, when the layer of precipitated proteid will 
have settled to the bottom. The graduations indi- 
cate the grams of proteid in the liter. If the 
amount of proteid is large, it is advisable to 
dilute the urine with two or more parts of dis- 
tilled water before beginning the test, and then to 
correct the final reading by the dilution. To 
insure accuracy the tube should stand in a tem- 
perature of about 15° C. (60° F.). Amounts 
of albumin less than 0.05 per cent, will not sedi- 
ment, so cannot be estimated by this method. 

Modification of the Esbach Test. — The great 
drawback has always been the necessity of wait- 
ing twenty-four hours for complete precipitation before read- 
ing the result. This method differs from Esbach's simply in 
the addition of 10 drops of a' 10 per cent, ferric chlorid solu- 
tion to the measured amount of urine after it is put in the tube 
andi before the Esbach solution is added, and after gentle mix- 
ing place the tube in a water bath at a temperature of 72° C. 
The precipitation begins almosii immediately and is complete in 
a few minutes, when the results are read in the usual manner. 
This method is but slightly more complicated than the usual 
Esbach estimation, and has been employed by the author in a 
series of specimens, using the old method for control. N'o 




ALBUMIN. 325 

differences were noted. It seems to be an accurate modification 
of much value in saving time. 

Tsuchiya's Modification of Esbacli Method. — ^First render 
nrine acid with a few drops of acetic acid to prevent bubbling. 
Fill the Esbach tube .with urine to the mark U, then add re- 
agent (see Appendix, page 496), to the mark E. Cork the tube 
and invert several times to thoroughly mix the urine and re- 
agent. (Do not shake.) The precipitate is allowed to settle 
by maintaining the tube in an upright position at room tem- 
perature for twenty-four hours. The height of the precipitate 
is then read by the scale on the tube. This figure gives the 
gTams of albumen per liter. 

Caution". — ^Specimens containing over 5 grams per liter 
should be diluted with water before testing, in order to keep 
the amount of albumen below this figure in the test tube. 
Large quantities introduce an element of error. 

The advantages of this test over the original Esbach are 
said to be: 

(1) The precipitate rarely floats. 

(2) The precipitate settles more evenly. 

(3) The readings are but slightly affected by temperature. 

(4) The element of error is not over 0.3 grams per liter. 

(5) The reagent is non-staining. 

Albumosuria. — Albumose appears in the urine as a part 
product of proteid metabolism. It is a pre-peptone which ap- 
pears in the blood and can be produced by^ artificial digestion. 
Pathologically, its continued and marked presence in the urine 
usually denotes multiple myelomata. It has also been noted in 
syphilis, pneumonia, peritonitis, and cholera. 

The Test. — Add concentrated nitric acid to the urine, agi- 
tate, and allow to stand in a cool place. A heavy precipitate will 
occur, which upon heating disappears, only to reappear on cool- 
ing. At the same time the mixture gradually assumes an 
intense yellow color. 

Albumin of Bence-Jones. — This substance has been repeat- 
edly found in cases of multiple myelitis. Its exact nature is 
not definitely known, but it is generally regarded as an albumose. 
Its reaction to heat and nitric acid^ is similar to that of albu- 
mose. The test for its positive identification is complicated and 



326 THE URINE. 

difficulty and will hardly be required of the clinical laboratory 
worker. 

Nucleo-Albumin. — Nncleo-albumin, beyond a faint trace^ 
will give the same reactions as true blood-albumins. Its pres- 
ence may be inferred when there is an albumin reaction in a 
specimen which shows an absence of renal elements with an 
excess of epithelia of bladder and urethral type. 

Test. — To dilute acid urine add an excess of acetic acid, 
stand aside, and gradually a faint cloud will appear. 

DiFFEEEiS'TiAL Test. — If the preceding! test gives a positive 
reaction, take a fresh portion of the same specimen, and first add 
one-sixth volume of a saturated sodium chlorid solution, and 
then some dilute acetic acid. The presence of sodium chlorid 
will prevent the precipitation of nucleo-albumin in the last test. 

Fibrin, when present in the urine, usually occurs in suf- 
ficient amounts to form clots, which are easily distinguishable. 
When the amount is small it may become necessary to determine 
the presence of this substance by chemical means. 

Test for Serum-globulin. — Add to some urine contained 
in a small beaker sufficient ammonia water to produce a slightly 
alkaline reaction; a cloud of phosphates will appear and will 
settle to the bottom. As soon as a layer of clear urine appears, 
decant this into a test-tube. Mix this clear urine with an equal 
quantity of a clear saturated solution of ammonium sulphate. 
If a flocculent precipitate appears in a few minutes the albumin 
present is serum-globulin. 

Test for Serum-albumin. — Take the final solution ob- 
tained in the foregoing test and filter it free from serum-globu- 
lin. Heat the filtrate to boiling, and add one-tenth volume of 
strong nitric acid. If a precipitate now appears the urine con- 
tains serum-albumin. 

Proteose. — ^Mix about 50 cubic centimeters of urine con- 
tained in a beaker with 5 cubic centimeters of strong nitric 
acid. Heat the solution rapidly to about 80° C. If a pre- 
cipitate appears, prepare a hot filter-paper by pouring boiling 
water through it in a funnel. Now filter the hot mixture of 
urine and nitric acid. Add to the clear, hot filtrate its ovno. 
volume of a clear, saturated solution of sodium chlorid. Allow 



GLUCOSE. 327 

the mixture to j cool. If any precipitate appears in this cooled 
mixture then proteose was present in the specimen of urine 
under examination. 

GLUCOSE. 

General Considerations. — ^A trace of sugar can usually be 
detected in apparently normal urine from healthy subjects by 
special tests of great delicacy. The ordinary tests employed in 
clinical medicine, however, do not react to these minute 
amounts of sugar, hence the findings of glucose by the tests 
about to be outlined must, in every case, be considered 
pathologic. 

The reducing power of normal urine is, in part, due to the 
activity of uric acid and creatinin, and is equal to about a 0.1 
per cent, solution of glucose. 

Under pathologic conditions the reducing power of urine 
may be enormously increased, due to the abnormal presence of 
glucose. This may be present up to as much as fifty grains to 
the ounce (10 per cent.). 

The detection of sugar in the urine is based upon a knowl- 
edge of the following properties of glucose: 

1. In hot alkaline solutions, reduces the oxides of copper 
and bismuth. 

2. With brewer^s yeast, ferments, forming alcohol and car- 
bon dioxid. 

3. Enters into chemical combination with phenylhydrazln 
to formi characteristic insoluble crystals of glucosazone. 

4. Deflects polarized light to the right. 

In the application of all tests but the fermentation-test, it 
is necessary to use urine free from albumin. If this be present 
it must first be removed by the addition of dilute acetic acid and 
boiling to cause precipitation. This is then filtered out, and the 
filtrate used in the tests for sugar. 

Reduction Tests. — ^Megchod of Fehling^ (for preparation 
of reagent see Appendix) : The sample of urine to be tested is 
first examined carefully for the presence of albumin. If this be 

3 The suggestions for the performaxice of this test are taken from a com- 
munication by C. W. Louis Hacker, M.D., appearing in the Jour. Amer. Med. 
Assoc, page 252, Jan. 25, 1908. 



328 THE UEINE. 

found it must be removed by heat and acetic acid, and the filtrate 
used for the test. About 4 cubic centimeters of freshly pre- 
pared Fehling's solution are diluted with three parts of water, 
and brought just to the boiling point in a clean test-tube. 
Immediately two drops of proteid-free urine are added, and the 
tube shaken vigorousl}^ If the urine contains more than 2 per 
cent, of sugar, a yellowish-red precipitate immediately appears, 
changing rapidly to a brownish-red or bright red, which settles 
out slowly, leaving a greenish-blue supernatant fluid. If the 
reaction be positive, it is better to roughly dilute the urine with 
two or four parts of water, and to perform the test again. 

With the strengths of dextrose from % "to 2' P^r cent., two 
drops of urine, as stated above, will cause a characteristic pre- 
cipitation of red suboxid of copper, about which there can be 
no doubt. With smaller percentages the change may come slowly, 
but even then heat need not be applied after the addition of the 
urine. Here little change may be seen in the Fehling's solution 
by transmitted light, but by reflected Hght a brownish-red tint 
is evident, which finally develops into a decided light-red pre- 
cipitate, gradually settling out and leaving a clear, blue, super- 
natant fluid. The pentoses, lactose, and even maltose, by this 
method, do not give the characteristic reaction. Urines showing 
this reaction always give a reaction with Nylander's solution 
(see page 330). 

If no change occurs in the Fehling's solution after the addi- 
tion of the two drops as above, the solution may then be warmed 
just to the boiling point, and froni two to four drops of urine 
added and the mixture observed. This should be repeated until 
in all twenty drops of urine have been added. If no change 
occurs at this point, the urine does not contain dextrose to the 
extent of 0.5 per cent. 

Cautions. — The disadvantages of using large amounts of 
urine (an equal or even one-half volume of the Fehling's solu- 
tion) are two-fold. In the first place, the strongly alkaHne cop- 
per solution with urine of high specific gravity throws down a 
more or less voluminous precipitate of cupric phosphate, which 
takes out the blue color of the solution, and changes it to a dirty, 
dark green. In the second place, the alkaline solution, acting on 
the ammonium salts in the urine, liberates sufficient ammonia to 



GLUCOSE. 329 

hold in solution small amounts of the red suboxide of copper, 
obviously interfering with the detection of traces of dextrose. 
Very frequently under these conditions, and especially if the 
solution has been boiled, even for a few seconds after the addi- 
tion of the urine, the greenish solution slowly takes on a dirty, 
yellow-brown color, then become turbid, and, eventually, on 
standing, there separates out a finely divided opalescent, green- 
ish-yellow precipitate, which only settles out completely after the 
tube has been allowed to stand overnight. In certain cases this 
reaction does not begin until a few seconds have elapsed after 
the addition of the urine. Such a reaction may be termed a 
pseudo-reaction. 

Fallacies. — Neglect of the above precautions will result in 
the occurrence of the pseudo-reaction in about 5 per cent, of nor- 
mal urines examined. 

The following substances, if present in the urine in more 
than normal amounts, will result in partial reduction of Feh- 
ling^s solution. Uric acid, creatinin, hippuric acid, mucin, 
hypoxanthin, and alkapton. Also the presence of the oxidation 
products of the following drugs : The alkaloids, arsenic, carbolic 
acid, and hexamethylenamin (urotropin). The latter substance 
does not ordinarily reduce bismuth. 

De Jager's Test.4 — ^De Jager describes the following as 
an improvement in his older test for sugar in the urine: Milk 
of lime, made by adding 30 grams of calcium hydroxide to water, 
up to 100 cubic centimeters, is allowed to stand at least twenty- 
four hours before it is employed in the test. To 5 cubic centi- 
meters of the urine add 10 drops of milk of lime and 5 drops of 
a 10 per cent, solution of copper sulphate. The mixture is then 
heated to boiling. Where sugar is present, a red or violet pre- 
cipitate appears, unless the amount of sugar is very la,rge, when a 
yellow color is seen. The limit of sensitiveness of the test is 
1 part of sugar in 10,000. The lime suspension should always 
be well shaken before it is used. 

Haines's Test. — Haines has introduced a modification of 
Trommer's test by adding glycerin instead of Eochelle salt, to 
increase the amount of copper in solution. This test is much 
more convenient than Fehling's, the solution having the advan- 

4 Zentralblatt fur innere Medizin, June 22, 1912. 



330 THE URINE. 

tage of keeping almost indefinitely. It is, however, far less deli- 
cate than is Benedict's and is reduced by preservatives as well 
as by excess of many normal nrinary constituents, especially by 
uric acid and creatinin. 

To test, one or two cubic centimeters of this solution (see 
Appendix) are placed in a test-tube and gently boiled. Six drops 
of the suspected urine are added and the upper portion of the 
mixture brought to a boil and immediately removed from the 
flame. If sugar be present, an abundant yellow or yellowish-red 
precipitate is thrown down; if no such precipitate occurs, sugar 
is absent. 

Caution. — ^The precautions to be observed in using this test 
are : never to add a total of more than 10 drops of urine and not 
to boil the mixture for more than one or two seconds after the 
addition of the urine. 

Trommer's Test. — To half -inch of urine in a test-tube, add 
one inch of liquor potassii and a few drops of a 10 per cent, solu- 
tion of copper sulphate sufficient to cause blue discoloration with 
slight cloudiness. Heat this mixture to the first signs of boil- 
ing. If sugar be present the copper will be reduced to yellow or 
red oxid. Slight precipitations, which may occur after pro- 
longed boiling, are no proof of sugar. 

Delicacy. — This test, if carefully performed, will demon- 
strate the presence of sugar in 0.025 per cent, solution. 

BoTTGER^s Bismuth Test. — Urine containing coagulable 
proteids must first have them removed by heat and acetic acid. 
Mix in a clean test-tube equal volumes of urine and liquor po- 
tassii, and add a few grains of bismuth subnitrate and boil. In 
the presence of glucose the white powder in the bottom of the 
tube will gradually become gray, brown, or black. 

Nylander's Bismuth Test. — Nylander modifies the pre- 
ceding test by substituting for the subnitrate a special reagent 
containing bismuth (for reagent see Appendix). Albumin, if 
present in the urine, must be removed in the usual way. To 
10 cubic centimeters of Nylander's reagent, add 1 cubic centi- 
meter of urine and bring to the boiling point. A brown or 
black discoloration of the liquid denotes sugar. 

Fallacies of bismuth tests. A slight reduction of bismuth 
may be caused by turpentine, eucalyptus, or the ingestion of large 



GLUCOSE. 331 

amounts of quinine. Albumin and sulphur compounds in urine 
produce a black precipitate. 

Delicacy. — These tests show the presence of glucose in 
0.025 per cent, solution. 

Phentlhydkazin Test. — This test depends upon the pro- 
duction of characteristic crystals of phenylglucosazone by the 
combination of glucose and phenylhydrazin in hot solution, and 
their recognition with the aid of a microscope. 

The Test. — Fill a beaker half full of water and warm upon 
the wire-gauze over a low OBunsen flame. Place a test-tube, con- 
taining the urine, which has been acidulated with a few drops of 
dilute acetic acid, in the beaker. While waiting for this to 
warm prepare the following solution (this must be made fresh, 
as it does not keep well) : Weigh out 1 gram of phenylhy- 
drazin hydrochlorid and 2 grams of sodium acetate. Mix the 
salts and dissolve in 10 cubic centimeters of distilled water 
acidulated with 2 drops of dilute acetic acid. Filter to clarify. 
After the water in the beaker has boiled for five minutes, observe 
the urine in the tube; if it has become turbid, filter. To 10 
cubic centimeters of hot urine in a clean test-tube, add 5 cubic 
centimeters of the filtered reagent, replace in a beaker of boiling 
water, and continue boiling for one hour. Then cool quickly by 
immersing the tube in cold water. If sugar was present in the 
urine a crystalline precipitate will appear. This should be taken 
up in a pipette and transferred to a microscope slide for exami- 
nation. The characteristic crj^stals of phenylglucosazone are 
fine, faintly yellow needles arranged in the forms of fans, 
rosettes, and sheaves. 

Fallacy. — Levulose, if present in the urine, will produce 
similar crystals; these can be differentiated by a comparison of 
the fusing points of the two compounds. 

Phenylglucosazone fuses at 204° C. 

Phenyllevulosazone fuses at 150° C. 

Delicacy. — Glucose forms characteristic crystals when pres- 
ent in 0.025 per cent, solution. 



332 THE URINE. 

QUANTITATIVE OR VOLUMETRIC DETERMINATIONS 
OF GLUCOSE IN THE URINE. 

Method of Fehlixg. — Measure into a beaker or porcelain 
dish 10 cubic centimeters of accurately prepared Fehling's solu- 
tion and 40 cubic centimeters of distilled water. Heat the mix- 
ture over wire-gauze to boiling. Prepare a dilute solution of 
suspected urine by adding 9 parts of distilled water to 1 
part of urine. This mixture is placed in a burette. Now to the 
boiling solution add the dilute urine, a few drops at a time, from 
the burette. Continue adding, boiling, and stirring until the 
blue color of the Fehling solution completely disappears when 
viewed by transmitted light. ISTote accurately the number of 
cubic centimeters of urine used. 

Fehling^s solution is a standard solution of copper sulphate, 
10 cubic centimeters of which is exactly decolorized by 0.05 
gram of glucose. 

Example. — Suppose 8 cubic centimeters of diluted urine 
were used in decomposing 10 cubic centimeters of Fehling^s 
solution. Then, of undiluted urine, 0.88 cubic centimeter was 
required. This amount then contained 0.05 gram (10 x 0.005) 
glucose. To calculate the percentage of sugar in the sample 
of urine: 0.8 : 0.05 : : 100: X, which, in this case, "X" equals 
6.25 per cent. To calculate the grams of glucose voided in 
twenty-four hours: If 1500 cubic centimeters of urine were 
voided in twenty-four hours, then 0.8 : 0.05 : : 1500 : X, which 
equals 93.75 grams glucose. 

Caution. — ^The dilute urine should be added very slowly 
with frequent boilings and examinations by transmitted light to 
avoid passing the end reaction. 

Fallacies. — The sources of error in this test are the same as 
those given under Fehling^s qualitative test on page 329. 

Benedict's Second Method. — This newer method of 
Benedict is to be preferred as it employs but one solution which 
keeps well. The reagent is far more sensitive (about 10 times) 
to glucose than the commonly used copper solutions; moreover 
it is not appreciably reduced by uric acid or by creatinin nor by 
such preservatives as chloral, chloroform or formaldehyde. 

Technic. — Accurately measure 25.0 cubic centimeters of the 
reagent into a porcelain dish and add 5 to 8 grams of anhydrous 



DETERMINATION OF GLUCOSE IN THE URINE. 333 

sodium carbonate and a small quantity of powdered piimace 
stone. Boil vigorously for a few moments and then run in from 
a burette the specimen (diluted 1 : 10 unless the sugar content 
is known to be slight), until a whitq precipitate is formed and 
the blue color is distinctly diminished. Then continue the addi- 
tion more slowly, boiling the reagent vigorously until the last 
trace of blue has disappeared, this marks the end point. The 
amount of urine (plain or diluted) used as read from the 
burette is used to calculate the sugar percentage as in the pre- 
ceding methods. 

Bang^s Method. — The principle of this method is as fol- 
lows: The urine is treated with an excess of standard alkaline 
copper solution (see Appendix, page 495) and boiled to exhaust 
the glucose present by reducing part of the copper content. The 
amount of copper remaining in excess is then determined by 
titration with a solution of hydroxylamin sulphate, and from this 
the amount of sugar is calculated. (For reagent, see Appendix.) 

Teclmic. — Ten cubic centimeters of urine (5 or 2 cubic 
centimeters diluted with water to 10 cubic centimeters if more 
than 0.6 per cent, of sugar is present) are measured into a 200 
cubic centimeter Jena-Erlenmeyer flask and treated with 50 
cubic centimeters of the copper solution. Heat on a wire gauze to 
boiling for exactly three minutes. Cool quickly to room temper- 
ature by immersion of the flask in cool water. Now titrate with 
the hydroxylamin solution until a colorless mixture results. 
Prom the number of cubic centimeters of hydroxylamin sulphate 
solution used, calculate the sugar in milligrams by means of the 
table. A simple calculation yields the percentage and total 
amounts. (See Appendix for table, page 510.) 

Purdy's Quantitative Method. — ^Into' a beaker or boiling 
flask of 250 cubic centimeters^ capacity put 35 cubic centimeters 
of Purdy's reagent (see Appendix) and 70 cubic centimeters of 
distilled water. Boil steadily over a wire gauze, and add urine 
slowly from a burette until the blue color begins to fade ; now 
proceed cautiously, and after the addition of each drop wait for 
a few seconds to see if the end-reaction is complete. Continue 
adding until the solution of the reagent is colorless and trans- 
parent. To obtain tliis result the total amount of urine em- 
ployed must have contained 0.02 gram of glucose. 



334 THE URINE. 

Example. — Suppose the amoimt of urine employed to com- 
pletely decolorize the reagent was 4.5 cubic centimeters, then 
4.5 cubic centimeters of urine contained 0.02 gram of glucose; 
or 1 cubic centimeter of urine contains 0.0044 gram of glucose. 
This multiplied by 100 will give the percentage of glucose which 
is equal to 0.44. If the color of the reagent is changed by less 
than 4 cubic centimeters of urine, it is best to dilute the urine 
with one or two parts of water, and then multiply by this factor 
to obtain the final result. 

This method is rapid, the technic is simple, and the end- 
reaction definite and sharp. The reagent is stable. 

Fermentation Saccharimeter. — Xone of the substances found 
in the urine, which give the reduction reactions resembling glu- 
cose, are susceptible of alcoholic fermentation. This fact estab- 
lishes tlie accuracy of the test. The test itself depends upon the 
fact that when sugar-containing urine is mixed with a quantity 
of brewer's yeast and kept in a warm place for t^vent}^-four hours, 
it will ferment, giving off bubbles of 002? ^^tl at the same time 
forming alcohol. 

By measuring the amount of carbon dioxid gas formed 
during this fermentation process, we are enabled to estimate the 
percentage and amount of sugar contained in the specimen un- 
der examination. A specially shaped and graduated tube (see 
Fig. 63) is usually employed in this test. It is known as the 
Einhorn saccharimeter. The upright tube is graduated from its 
closed upper end downward from 0.1 to 1 per cent. It will not 
estimate sugar in amounts less than 0.1 per cent., and owing to 
the varying solubility of the various gases of the urine, depend- 
ing on differing reactions and the density, it cannot be regarded 
as absolutely accurate. 

The Test. — Urines containing less than 5 per cent, of 
glucose must be diluted five times with water. Urines contain- 
ing more than 5 per cent, must be diluted ten times before pro- 
ceeding with the test. 

Mix in a large test-tube about 20 cubic centimeters of 
properly diluted urine with one-twelfth of a cake of fresh 
compressed yeast. Completely fill the upright tube of the 
saccharimeter with this mixture, so as to exclude all air from 



DETERMINATION OF GLUCOSE IN THE URINE. 



335 



the graduated tube. Fill a second saccharimeter with yeast 
dissolved in distilled water or with nrine known to be free from 
sugar. These tubes are to be kept in a warm place and 
allowed to remain undisturbed for from eighteen to twenty- 




A B 

Fig. 63— Einhorn Saccharimeter in Use. 

A shows Gas formed after Twenty-four Hours from a Diabetic Urine (Dilution 

1 to 10) containing 2.7 Per Cent, of Sugar; B, Control with Normal Urine. 



four hours. By this time the sugar-containing urine will be 
found to have been displaced by gas in the vertical tube. The 
percentage corresponding to the level of the fluid will represent 
the percentage of sugar in the diluted urine. This figure, mul- 
tiplied by the dilution, will represent the percentage of sugar 
in the specimen under examination. 



336 THE URINE. 

Cautions. — The urme miist be faintly acid, and must be 
so diluted that the specific gravity will be less than 1008, and 
when diluted must contain less than 1 per cent, of glucose. If 
any gas is liberated by the yeast or fluid in the control-test, this 
amount must be subtracted from that indicated in the test-mix- 
ture before computing the percentage. 

Lohnstein's Saccliarimeter. — This instrument has much to 
recommend it over the older instruments. 

Technic. — Twelve cubic centimeters of mercury are placed 
in the bulb of the apparatus. One-half cubic centimeter of the 
urine to be tested is then floated upon the mercury and treated 
with a thick paste of compressed yeast diluted two or three 
times with water. The stopper is then carefully greased with 
vaseline and inserted so that the apertures in the instrument and 
in the stopper correspond. By tilting the apparatus a trifle the 
column of mercury in the long tube may be adjusted to the zero 
point of the scale. When this is done the stopper is turned so 
that the holes no longer correspond and the weight is placed on 
the stopper to prevent leakage from the increased pressure as 
gas is liberated during the fermentation. The apparatus is then 
placed in the incubator for six to eight hours and the extent of 
fermentation read off on the scale at the point to which the 
column of mercury has risen in the long arm of the instrument. 
After removing the apparatus from the incubator it is allowed 
to stand in the air for a few minutes, in order to cool as the 
scale is graduated to room temperature. 

This method gives results which correspond very closely to 
those of titration. 

The internal administration of mercuric chlorid, iodoform, 
salicylic acid, hexamethylamin, quinine, and other antiseptic 
drugs will inhibit fermentation, and therefore must be excluded 
before testing. 

Robert's Differential Density Method. — This method de- 
pends for its result upon the alteration in density occurring from 
the fermentation of saccharine urine. 

The Test. — Mix thoroughly about 120 cubic centimeters 
of urine with half a cake of compressed yeast. Take and 
record accurately the specific gravity of this mixture. Set aside 



POLARIMETRIC METHOD. 337 

in a warm place (thermostat preferred) for twenty-four hours, 
and at the expiration of this time again take the specific gravity 
and subtract the second reading from the first. Each degree 
of difference (showing density lost) represents approximately 
one .grain of sugar to the ounce. To obtain the percentage of 
sugar in the specimen, multiply the degrees of density lost 
through fermentation by the factor 0.219. 

Example. — Specific gravity before fermentation .... 1041 
Specific gravity after fermentation 1023 

Degrees of density lost 18 

This is approximately the grains of glucose in each ounce of urine; 
18 X 0.219 equals 3.94% glucose. 

This test is easily performed, and is conclusive evidence of 
the presence of glucose. It is, however, not strictly accurate, and 
is not to be relied upon when the amount of glucose present is 
less than 0.5 per cent. 

POLARISCOPIC DETERMINATION. 

The polariscope is an expensive instrument and for this 
reason it is not as generally employed for sugar determinations 
as the rapidity and ease of its use would seem to warrant. For 
clinical use the instrument is supplied with two special tubes, 
94.7 millimeters and 189.4 millimeters long, which permit a 
direct percentage reading of glucose; the short tube is used 
with dark, highly colored urines, the readings obtained being 
divided by two. The tubes must be perfectly clean and dry 
before using; hot water or other fiuid should not come in con- 
tact with them, since the expansion of the glass against the 
outer brass tubing may crack the former. 

Principle of Polarimetry. — ^An ordinary ray of light vibrates 
in every direction. If such a ray is caused to pass through a 
"polarizing'^ Nicol prism it is resolved into two rays, one of 
which vibrates in every direction as before and a second ray 
which vibrates in one plane only. This latter ray is said to be 
polarized. Many organic substances (sugar, proteins, etc.) 
have the power of twisting or rotating this plane of polarized 
light, the extent to which the plane is rotated depending upon 

22 



338 THE URINE. 

the number of molecules through which the polarized light 
passes. Substances which possess this power are said to be 
"optically active." The specific rotation of a substance is the 
rotation expressed in degrees which is afforded by 1 gram of 
substance dissolved in 1 cubic centimeter of water in a, tube 1 
decimeter in length. The specific rotation (a ) D, may be cal- 
culated by means of the following formula : 

a 
(a) D = 

in which 

D r= sodium light. 

a = observed rotation in degrees. 

p = grams of substance dissolved in 1 cubic centimeter 

liquid. 
1 = leng-th of the tube in decimeters. 
If the specific rotation has been determined and it is 
desired to ascertain the per cent, of the substance in solution, 
this may be obtained by the use of the following formula. 

a 

P = 

(a) Dl 

The value of p multiplied by 100 will be the percentage of 
the substance in solution. 

An instrument by means of which the extent of the rota- 
tion may be determined is called a polariscope or polarimeter. 
Such an instrument designed especially for the examination of 
sugar solutions is termed a saccharimeter or polarizing sac- 
charimeter. The polariscope illustrated, consists essentially 
of a long barrel provided with a Nicol prism at either end 
(Eig. 64). The solution under examination is contained in a 
tube which is placed between these two prisms. At the front end 
of the instrument is an adjusting eyepiece for focusing and a 
large recording disc which registers in degrees and fractions of 
a degree. The light is admitted into the far end of the instru- 
ment and is polarized by passing through a l^icol prism. This 
polarized ray then traverses the column of liquid within the 
tube mentioned above and if the substance is optically active the 
plane of the polarized ray is rotated to the right or left. Bodies 



POLAKISCOPIC DETERMINATION. 



339 



rotating the ray to the right are called dextro-rotatory and those 
rotating to the left levo-rotatory. 

The difference between this reading axid zero is a or the 
observed rotation in degrees. 

Detemiination of Sugar in 
Urine. — Polarizing saccharim- 
eters are also constructed by which 
the percentage of sugar in solu- 
tion is determined by making an 
observation, and multiplying the 
value of each division on a hori- 
zontal sliding scale by the value 
of the division expressed in terms 
of dextrose. This factor may 
vary according to the instrument. 

In this test the t^venty-four 
hours^ specimen is employed. 
The urine must be absolutely 
clear and must contain no albu- 
min. Moreover, it is essential 
that such substances as giycu- 
ronic acid and B-oxybutyric acid 
be removed before polarization 
as they will introduce consider- 
able error in the reading. The 
best clearing agent is shaking 
the urine with HCl and blood 
charcoal and filtering. Instead 
of this method, the urine, acidi- 
fied with acetic acid may be 
treated with a solution of nor- 
mal lead acetate, which will 
precipitate the albumin and re- 
move excess of pigment. The mixture is then filtered and the 
clear filtrate is used for the test. It must be remembered that 
a correction must be made for all additions in the percentage 
of sugar in the filtrate. If the urine be cleared with a solution 
of lead acetate, this may be done by adding a measured quantity 
of solution to a measured amount of urine, for example 25 cubic 




Fig. 64.— 
Ultzmann's Polariscope. 



340 THE URINE. 

centimeters of a 10 per cent, solution of lead acetate to 75 cubic 
centimeters of urine. With this mixture the sugar in the filtrate 
will represent only three-fourths of that of the original urine. 
Basic lead acetate should not be used as it precipitates various 
sugars. If the urine contains no albumin, magnesium oxid or 
silicic acid may be used as clearing agents. 

The clear urine is now filled into the polariscope tube 
(189.4 millimeters in leng*th) until the fluid is convex above 
the end of the tube. The glass disc is then placed over the end 
of the tube and secured in place by screwing down the metal 
cap. Air bubbles must be avoided, since their presence makes a 
satisfactorj'- reading impossible. The tube is next placed in the 
polariscope. 

The examination must be made in a dark room with a 
sodium flame for illumination. After focusing, readings are 
made, first without the urine, to determine whether the zero 
point is accurate; next after refocusing, with the tube of urine; 
starting at zero, the handle is rotated until the entire field is 
equally illuminated. At least six readings should be made. The 
percentage is read directly from the scale, tenths being obtained 
on the vernier. (In case the instrument is supplied only with 
the standard tubes of 100- and 200- millimeter lengths, the per- 
centage of glucose may be calculated from the polariscopic read- 
ings by dividing the results by 0.527.) 

The method gives fairly satisfactory results. When no dis- 
turbing bodies are present in the urine, the error ,is about 0.1 
per cent, of glucose. 

Sources of Error. — (1) Albumin, when present, must be 
removed before making polariscopic determination of glucose, 
otherwise the albumin, which is levo-rotatory, will counter- 
balance the dextro-rotatory glucose, in part at least. (2) Alka- 
linity of the urine precludes its use with the polariscope, since 
it has been shown that in alkaline media dextrose may be con- 
verted into levulose. The addition of a preservative to a speci- 
men usually suffices to prevent an acid urine becoming alkaline. 
(3) B-oxybutyric acid is levo-rotatory, and its presence, there- 
fore, interferes with the accurate estimation of glucose. (4) 
The combined gl3^curonates are levo-rotatory, though they are 
generally present in such small quantity as to produce only 



POLAEISCOPIC DETERMINATION. 34I 

slight rotation of the polarized light. (5) Levulose, when pres- 
ent in the nrine with glucose, is antagonistic, and lowers the 
reading for glucose. (6) Maltose is occasionally present in the 
urine with glucose. Since it is more powerfully dextro-rotatory 
than glucose, the reading may give a value which is too high. 

Clinical Significance of Glycosuria. — The presence of glu- 
cose in the urine is pathologic. If the amount be abundant and 
persists, associated with copious water-drinking and eating, while 
the patient is at the same time emaciating, it is indicative of 
diabetes mellitus. The urine in this condition is usually pale 
with a fruity odor, and while over-abundant, has a high specific 
gravity. As much as 10,000 cubic centimeters of urine may be 
excreted in twenty-four hours; at the same time the specific 
gravity may be maintained at 1050. 

Temporary Glycosuria. — The appearance of small amounts 
of sugar may occur transiently in the urine after excessive inges- 
tion of sugar; and reducing substances, such as glycuronic acid, 
may give a spurious positive reaction. Copper reduction by such 
substances is not corroborated by either fermentation or the 
phenylhydrazin tests. 

Glycosuria also may accompany certain diseases of the 
brain, spinal cord, and pancreas, or be a transitory accompani- 
ment of phthisis, pneumonia, cirrhosis, pulmonary edema, or 
cholera. 

LEVULOSE (D-FRUCTOSE). 

Levulose is found very widely distributed throughout the 
vegetable kingdom, especially in fruits. Honey is almost a pure 
levulose. It may be found in the urine, transudates, or exudates 
after a large intake of levulose-containing food or may occur 
spontaneously, even when the subject has taken little such food. 

Levulose forms exactly the same phenylosazon as does glu- 
cose, so that it is a matter of great difficulty to differentiate those 
bodies by the ordinary tests (see page 331). 

Seliwanoff's Test. — This test has been advanced as one 
characteristic for the ketones in distinction from the aldeliydes. 
Ten cubic centimeters of urine are treated with a few crystals 
of resorcin and 5 cubic centimeters of concentrated HCl. If the 
mixture be warmed, a brilliant red color appears in the presence 



342 THE URINE. 

of a ketone (levulose)^ while no coloration is observed with an 
aldehyd (glucose). If the mixture be heated too strongly or too 
long, mannose and maltose may also give a positive test. 

If the red solution formed in this reaction be neutralized 
with sodium carbonate and extracted with amyl alcohol or, prefer- 
ably, with acetic ether (Borchardt), the extract will have a yellow 
color with a faint green fluorescence which becomes rose-red on 
the addition of alcohol. The spectrum of this solution shows a 
sharp line in the green between E and b, while if the solution 
be quite concentrated a second, weaker line will be seen in the 
blue at F. 

LACTOSURIA. 

Milk sugar occurs occasionally in the urine of nursing 
women, particularly toward the end of lactation. It will reduce 
Fehling's and Nylander^s solutions, but returns a negative result 
with the phenylhydrazin test. It ferments very slowly. 

MALTOSURIA. 

Maltose has been found in the urine of diabetic patients, 
and when present reduces copper and ferments with yeast. Its 
occurrence is sufficiently rare to be disregarded in the clinical 
laboratory. 

PENTOSURIA. 

Diabetic and morphine habitues occasionally show in the 
urine traces of pentose. This will reduce copper, but will not 
ferment. 

Newmann's Orcin Test. — This test may be employed to dis- 
tinguish between the pentoses, the gl3^curonates, and dextrose. 

Technic. — Three cubic centimeters of urine are treated 
with 10 drops of a 5 per cent, alcoholic orcin solution and 10 
cubic centimeters of glacial acetic acid. The mixture is now 
brought to the boiling point and allowed to cool. After cooling 
concentrated sulphuric acid is added, drop by drop, and shaken 
until about 20 drops have been added. In the presence of 
pentoses the color becomes olive-green, glycuronates turn the 
solution violet, and dextrose gives a carmine color. 



ACETONE. 343 

ACETONE. 

Usually in advanced diabetics, sometimes in fever or in 
perfect health, particularly in patients whose diet is rich in pro- 
teids, the urine may have an ethereal odor, and will give posi- 
tive reactions to tests for acetone. 

Acetone is more constantly present in diphtheria and scarlet 
fever than in typhoid fever, and, according to recent authorities, 
it is the result of the reduction of food intake. 

In the course of a case of diabetes a decline in the percent- 
age of sugar, accompanied by lowered specific gravity, but with- 
out corresponding improvement in the general symptoms, may 
indicate impending coma. At this time acetone will be found 
in the urine in increasing amounts, with or without diacetic acid 
and oxybutyria acid. 

Ethylene-Dlamin-Hydrate Test. — This is the simplest and 
is probably the most satisfactory for clinical purposes. 

Test. — Place in a clean test-tube about 5 cubic centimeters 
of distilled water, and to this add about one grain of sodium 
nitro-prusside ; shake to effect solution. Add to this an equal 
amount of suspected urine and thoroughly mix. Gently overlay 
the mixture with a few drops of a 10 per cent, solution of 
ethylene-diamin-hydrate. A pink or ruby-red color at the zone 
of contact denotes the presence of acetone. A faint white cloud, 
which appears on the addition of the reagent, does not denote 
acetone. The solution of sodium nitro-prusside must be freshly 
prepared. 

Legal^s Test. — To 5 cubic centimeters of urine in a test- 
tube, add a few drops of a freshly prepared solution of sodium 
nitro-prusside and a few drops of sodium hydrate solution. A 
red color will appear in all urine, which soon changes to yellow. 
If this is now overlaid with strong acetic acid a change in color 
at the line of contact from yellow to carmine or purplish red 
indicates acetone. 

Frommer^s Test may be applied to the urine direct. This 
test is described by Dr. Jacob Eosenbloom^ as follows : — 

"About 10 cubic centimeters of urine are treated with about 
1 gram of sodium hydroxide in substance, and, without waiting 



5 Jour. A. M. A., August 10, 1912. 



344 THE URINE. 

for it to dissolve, 10 to 12 drops of a 10 per cent, solution of 
salicyl aldehyde in absolute alcohol are added. The mixture is 
heated to 70° C. In the presence of acetone a marked purple- 
red color develops at the zone of contact with the alkali. From- 
mer asserts that this test can indicate the presence of 0.000001 
gram acetone in 8 cubic centimeters of water." 

The urine should be diluted so that its specific gravity is 
reduced to about 1010; otherwise confusing colors occur that 
render interpretation difficult. When properly diluted, however, 
urine containing only minute or normal quantities of acetone will 
give after ten or fifteen minutes' standing only a straw or faintly 
pink color. 

Tests Requiring Distilled Urine. — Lieben's Test. — To a 
few cubic centimeters of the distillate of urine are added a few 
drops of concentrated sodium or potassium hydrate and a few 
drops of a solution of iodin in potassium iodid (Lugors). On 
slightly warming the mixture yellow cr)^stals of iodoform will 
separate, which may be recognized by their characteristic odor 
as well as by their hexagonal shape when examined under the 
microscope. This reaction is given by alcohol as well as by 
acetone but occurs more slowly. It will show amounts of acetone 
varying between 0.01 and 0.001 milligram per cubic centimeter. 

Gunning's Test. — This is a modification of the Lieben test 
and is much more specific, reacting only with acetone. To the 
distillate from the urine are added a few drops of an alcoholic 
solution of iodin and the mixture treated with ammonia until 
a black precipitate of nitrogen iodid forms. On allowing the 
tube to stand for periods varying between twelve and twenty-four 
hours, this black precipitate disappears, leaving a yellow sedi- 
ment of iodoform, which may be recognized as mentioned above. 
This test is less delicate than the original one of Lieben, detect- 
ing acetone when present in amounts of 0.01 milligram per 
cubic centimeter or more. 

Test for Aceto-acetic Acid. — ^W. H. Hurtley offers the 
following new test as being the most suitable to get an idea of the 
amount of aceto-acetic acid in a urine. To 10 cubic centimeters 
of urine add ^.5 cubic centimeters of concentrated hydrochloric 



DIACETIC ACID. 345 

acid and 1 cubic centimeter of a 1 per cent, solution of sodium 
nitrate; shake and allow the mixture to stand for two minutes. 
Add 15 cubic centimeters of strong ammonia, followed by 5 cubic 
centimeters of a 10 per cent, solution of ferrous sulphate or a 
ferrous chloride solution of equivalent iron content (2 grams 
iron in 100 cubic centimeters) ; shake; pour into a Nessler glass, 
and allow to stand undisturbed. Do not filter. A positive 
reaction is present when a beautiful violet or purple color is 
produced. The reaction is slow, the speed with which the color 
develops depending on the concentration of the aceto-acetic acid 
present. Acetone does not give this test. The test is delicate to 
the detection of 1 part of aceto-acetic acid in 50,000 parts of 
solution. 

DIACETIC ACID. 

Diacetic acid does not occur in the urine of healthy individ- 
uals upon an ordinary diet, except possibly in very small quan- 
tities. It has been observed pathologically, usually in combina- 
tion with ammonia, in severe forms of diabetes, in fevers, in 
metabolic disturbances and their coincident autointoxications, in 
gastric carcinoma, and in alcoholism. 

Since acetone is derived from diacetic acid and the condi- 
tions requisite for the formation of diacetic acid are identical 
with those for the formation of acetone, the two substances are 
almost always found in conjunction. If little diacetic acid is 
formed it is all transformed into acetone ; if much is formed then 
both substances will be found in the urine. 

Gbrhardt''s Diacetic Ferric Chlorid Eeaction. — Accord- 
ing to the customary directions in the text-books for the per- 
formance of the Gerhardt test for diacetic acid (aceto-acetic 
acid), on the addition of the ferric chlorid solution to the urine 
a marked and obscuring precipitate of phosphates is usually 
obtained. This may be avoided by the chemist's simple expedi- 
ent of adding the urine drop by drop to 10 to 15 cubic centi- 
meters of the ferric chlorid solution. ^ In tliis way it is also 
possible to get a rough idea of the amount of the aceto-acetic 
acid present. If this is present in quantity it will give a strong 
Bordeaux-red coloration with the addition of a few drops 

6 F. H. Church : Jour. A. M. A., Oct. 2, 1909. 



346 THE URINE. 

of the nrine; otherwise this coloration will not be present nntil 
1 or 2 cubic centimeters of the nrine has been added. Snlpho- 
cyanides, sodium acetate, salicylic acid, antipyrin, thalin and 
aromatic substances may produce a similar red color. For this 
reason the presence of diacetic acid should be assumed only after 
positive results have been obtained by the two following control 
tests" : — 

1. Boil the urine employed for the test and the red color 
should be very much fainter, because boiling gradually trans- 
forms diacetic acid into acetone. 2. The urine is acidified with 
sulphuric acid and then some ether is added. If this is shaken 
with a diluted solution of ferric chlorid the aqueous layer will 
turn red. 

OXYBUTYRIC ACID. 

This substance cannot be readily detected by chemical means 
unless a long and difficult preliminary process is resorted to in 
order to separate it from the other urinary constituents. These 
methods will be found in larger works on clinical and physio- 
logic chemistry. 

BILE-PIGMENTS. 

General Considerations. — Bile-pigments are never found in 
the urine under normal circumstances. As a rule the freshly 
voided urine only contains the oxidized derivative, bilirubin. If 
a cystitis should exist, then subsequent oxidation products, 
biliverdin, bilifuscin, biliprasin, and bilihumin, may also be 
encountered. 

Bile-containing urines present a more or less characteristic 
appearance ; their color may vary from a bright golden-yellow to 
a dark greenish-brown. On microscopic examination it is usual 
to find the morphologic elements of the urine stained yellow or 
reddish-brown. The same color may be imparted to the foam 
on shaking. 

Gmelin's Test for Bile-pigments. — Put some yellow nitric 
acid in a test-tube and gently overlay it with the suspected urine. 
In the presence of bile-pigment a play of colors will be observed 
at the zone of contact. Green will be seen nearest the urine and 



7 Sahli : "Diagnostic Methods," 1907. 



BILE-PIGMENTS. 347 

orange in the npper part of the nitric acid. This test is exceed- 
ingly sensitive, indicating the presence of bile-pigment in a dilu- 
tion of 1 to 80,000. 

Nitric Acid-Paper Test. — Moisten a piece of white filter or 
blotting paper with the suspected urine, and place it on a 
glazed tile or slab of porcelain. Allow one or two drops of 
commercial nitric acid to fall into the center of the wet paper. 
In the presence of bile-pigment concentric rings of blue, violet, 
green, and yellow will appear. A slight red reaction cannot be 
considered positive, as it may be produced by other substances 
than bile-pigment. 

Methylene-blub , Test. — A. Torday and A. Klier^ in 
working with the stomach contents of a jaundiced patient, found 
that the methylene-blue with which they were testing gave a 
green color instead of the usual blue. The urine of a jaundiced 
patient gave the same reaction. Investigating further, they 
found that various staining fluids gave similar reactions with 
the bile-stained urine. Pure bile-pigments have not been studied. 
The method consists of adding 1 drop of a 1 per cent, solution 
of the dye to 15 cubic centimeters of water, and to this 1 cubic 
centimeter of urine. Methylene-blue gives a green color; other 
stains give different reactions. Diazo positive urines also turn 
green with methylene-blue, but a much stronger solution of the 
stain is required than for bile. The delicacy of this test was 
found to be about twice as great as the iodine or the Gmelin test. 

Smith's Test. — This test has been described under other 
names, as those of Trosseau, Kathrein, Eosin, and Marechalt. A 
few cubic centimeters of urine, acidified if necessary with 
acetic acid, are treated with a 1 per cent, alcoholic solution of 
iodine in such a way that the latter solution is superimposed upon 
the urine, forming a distinct line of contact. If bilirubin or 
other biliary pigments be present, a beautiful emerald-green color 
is observed at the point of contact. This test is not very sensi- 
tive, indicating only 1 part of biliary pigment in 10,000 of 
urine. Certain drugs, especially antipyrin, may lead to the 
formation of a green color with this test. Thymol, if used as a 
preservative, may give rise to confusion with this and other 
bile tests. 

8 Deutsche med. V^och., August 19 and 26, 1909. 



348 THE URINE. 

Rosenbach's Test. — This is a modification of Gmelin's test 
and is performed as follows: A large quantity of urine which 
has been acidified with HCl is filtered several times through 
a thick filter-paper, which will hold back the bile-stained ele- 
ments of the urine. It is sometimes advisable to add a little milk 
of lime to the urine before filtering, instead of the HCl, as this 
will throw down the phosphates, which will carry with them the 
biliary pigments. If the filter-paper and contents be dried by 
pressing with a second dry filter-paper and a drop of yellow nitric 
acid allowed to fall upon H, distinct rings will be seen, which 
will be colored as in the nitric acid-paper test, the green one 
being external. 

Nakayama's Test. — This is a modification of the older 
Huppert test. Five cubic centimeters of acid urine are treated 
with an equal volume of 10 per cent, barium chlorid solution 
and the mixture centrifuged. The barium chlorid precipitates 
the phosphates and sulphates and carries down the biliary pig- 
ments. The supernatant fluid is then poured oft and 2 cubic 
centimeters of the following reagent are added to the precipitate. 
The reagent consists of 99 cubic centimeters of 95 per cent, 
alcohol, 1 cubic centimeter of concentrated HCl, and 0.4 gram 
of ferric chlorid. If this mixture of precipitate and reagent be 
heated to boiling, a bluish-green or a brilliant-green solution is 
obtained, which becomes violet or red on the addition of nitric 
acid. This test is said to indicate 1 part of bilirubin in 1,200,- 
000 parts of urine. This test is more complicated than Gmelin's 
and requires more time; yet the beautiful results obtained by it 
more than compensate for this difficulty. 

Import. — ^When bile is not freely and normally discharged 
from the bile-passages, the coloring matter from the retained 
bile is absorbed by the lymphatics, the various body tissues be- 
come stained with it, and it is partly eliminated in the urine. 

BILE ACIDS. 

Hay's Test. — This test depends upon the reduction of the 
surface tension of the urine in the presence of the bile acids. As 
advocated by Beddard and Pembrey, a pinch of powdered sulphur 
is sprinkled upon the surface of urine, which should be prefer- 
ably at a temperature not over 17° C. In normal urines the 



BILE ACIDS. 349 

sulphur will float upon the surface, while if the urine contains 
bile acids the sulphur may sink at once, indicating 1 part in 
10,000, or may sink only after a few seconds to one minute, 
thus indicating 1 part in 50,000.^ 

Oliver's Test. — This test is based upon the well-known 
property possessed by the bile acids of precipitating peptone 
when in acid solution. (For reagent see Appendix, page 504.) 

One to 2 cubic centimeters of clear filtered urine are 
placed in a test-tube and treated with 5 cubic centimeters of the 
reagent. In the presence of bile acids a decided milkiness appears 
at once, being the more intense, the larger the amount of bile 
acids. 

Pettenkofer's Test. — To the solution of the bile-salts (see 
foot-note) add, very slowly, two thirds of its volume of concen- 
trated sulphuric acid, so that the mixture does not become over- 
heated. Then add 3 to 5 drops of a solution of 1 part of cane 
sugar in 4 to 5 parts of water, and shake, whereupon the liquid 
turns a beautiful violet color. 

UROBILIN. 

General Consideratioiis. — Urobilin is closely allied to the 
normal urochrome, but is an abnormal product, probably result- 
ing from the action of reducing agents upon the normal urinary 
pigment. It is said to be identical with the stercobilin of the 
feces, and its behavior to chemical reagents can be tested by 
testing an alcoholic extract of feces. 

Test for Urobilin. — (a) Mix some urine in a test-tube with 
an equal amount of a 10 per cent, solution of zinc acetate. Filter 
off the precipitate, and the filtrate, in the presence of a demon- 
strable quantity of urobilin, will show a beautiful greenish 
fluorescence. With the spectroscope the urobilin in this solution 
gives a distinct absorption spectrum (see page 90). 

9 According to Salili, tMs test does not discriminate between biliary acids 
and biliary pigments, but clinically it is a matter of indifference wliieli are 
present. Phenol or aniline compounds lower the surface tension of the urine, 
so that their presence may lead to wrong conclusions. 

Note.— Hoppe-Seyler (Handbuch der Physiol, u. path.-chemischen Analyse, 
1893, p. 378) givesi the following directions for separation of bile-salts: Lead 
acetate and a very little ammonia are added to the urine. The resulting pre- 
cipitate is washed with water, then boiled with alcohol and filtered while hot. 
A few drops of 10 per cent, sodium hydrate are added to this solution, which is 
then evaporated over a water bath till dry, and the residue boiled out with 
absolute alcohol, in which the sodium salts of the bile-acids dissolve. The 
filtered alcoholic extract is evaporated to a small volume, when the resinous 
precipitate may be dissolved in a little water, and Pettenkofer's test applied. 



350 THE URINE. 

(b) Fill a clean test-tube three quarters full of urine, and 
add one drop of strong hydrochloric acid; to this add about one- 
sixth volume of aniylic alcohol, and after shaking slowly eight 
or ten times allow to separate by standing. Pour off the super- 
natant fluid and add to it thrice its volume of alcohol. Prepare 
separately a 5 per cent, solution of zinc chlorid, and add one 
drop of this to the alcoholic extract of the urine; finally add 
one drop of ammonium hydroxid. Zinc hydrate will be pre- 
cipitated, which should be filtered off. In the presence of 
urobilin the filtrate will present a beautiful green fluorescence. 

Significance. — Urobilin has been detected in cases of hepatic 
cirrhosis, malarial anemia, carcinoma, Addison's disease, and 
pancreatic disease with acholic stools. The estimation of uro- 
bilin gives a valuable indication as to the amount of blood being 
broken down within the body, and, therefore, serves as an early 
indication of the failure of the liver to functionate normally. 

HEMATURIA. 

General Considerations. — Blood may gain access to the 
urinary tract in many ways, and appear in the urine in varying 
amounts, from the smallest trace, demonstrable only by chem- 
ical means, to sufficient to produce bloody urine with clots. 

Blood from the kidney is usually well mixed with the urine, 
to which it imparts a brownish, smoky hue. Under the micro- 
scope tube-casts of blood-cells may be found. 

Blood from the ureter may be well mixed with the urine, 
or may appear in characteristic worm-like clots. 

Blood from the bladder may, or may not, show irregular 
clots. When recently shed it imparts a bright-red color to the 
urine; it is frequently accompanied by much mucus and large, 
flat epithelial cells in great numbers. 

Blood from the prostate, examined by the three-glass test, 
appears in the first and third glasses only. 

Blood from the urethra appears in the first glass, and is 
frequently clotted. 

Urine may show the presence of blood in the absence of 
any demonstrable lesion of the genito-urinary tract. It has 
been noted after the ingestion of strawberries, gooseberries, or 
a large amount of rhubarb. 



HEMATURIA. 351 

The blood may be contaminated from menstrual discharge. 
This possibility should always be borne in mind, and false con- 
elusions guarded against. 

Microscopic Appearance. — The most convenient method of 
demonstrating blood in the urine is by microscopic examination 
of the centrifugated or sedimentated sample. The corpuscles 
in ordinary acid urine maintain their characteristic bi-concave 
shape for a number of days, if decomposition and putrefaction 
are prevented. If the urine is of high specific gravity, the cells 
rapidly become crenated. On the other hand, if the urine be 
alkaline or becomes so after voiding, the corpuscles will appear 
swollen, shriveled, or shadowy. Urine containing more than a 
trace of blood is albuminous. 

Test for Occult Blood in the Urine. — To 10 cubic centi- 
meters of urine in a large test-tube, add about twice as much 
ether and agitate thoroughly by pouring from one test-tube to 
another several times. To this add a few grains of powdered 
gum guaiac, and again agitate. Next add 5 cubic centimeters 
of glacial acetic acid (99.4 per cent.) and again agitate; allow 
this to settle and then pour off the supernatant liquid and 
divide equally between two test-tubes; set one aside for a con- 
trol, and to the other add about 2 cubic centimeters of fresh 
hydrogen dioxid from a pipette, making an effort to have it 
settle to the bottom as a distinct layer. If a bluish discolora- 
tion appears either at the zone of contact or throughout the 
mixture, the presence of blood is demonstrated. 

A Simple Test for Blood in the Urine. i^ — About 10 cubic 
centimeters of urine are filtered. Before the last few drops 
of urine have left the filter, a little acetic acid is added and 
then a mixture of tincture of guaiac and ozonized turpentine 
poured on. A blue color appears almost at once, if blood be 
present. This modification is said to be far more sensitive than 
the original guaiac-turpentine method, even if the latter is 
used after extraction with ether. It is only in dilutions as high 
as 1 drop of blood to 10 liters of urine that the test fails. Practi- 
cally the only source of error is the presence of pus in the urine. 
The latter, however, does not interfere if the urine be boiled for 
a moment and cooled before subjecting it to the test. 

10 Wackers, Miinch. med. Wochen. 1911, No. 4. 



352 THE URINE. 

HEMOGLOBINURIA. 

This term signifies the presence of hemoglobin in the urine 
free from blood-corpuscles. Besides the foregoing test it may 
be demonstrated by means of the spectroscope. Faintly acid 
urine containing traces of hemoglobin will give two character- 
istic absorption bands of oxyhemoglobin. By the addition of a 
minute quantity of ammonium sulphid, the spectrum is changed 
to that of reduced hemoglobin. 

Hemoglobinuria occurs in a variety of conditions: scurvy, 
pyemia, purpura, typhus fever, poisoning from arsenic, phos- 
phorus, carbolic acid, chloral, and potassium chlorate. There 
has also been noted a periodic form of obscure origin. 

PYURIA. 

Pus, being an albuminous fluid, will cause urine containing 
more than a minute quantity to respond to the tests for albu- 
min. Urine containing much pus is turbid, and deposits on 
standing a white or greenish-white sediment which is insoluble 
in heat and in dilute mineral acids. The addition of hydrogen 
dioxid produces rapid effervescence. 

The sudden appearance of pus sediment in the urine sug- 
gests the rupture of an abscess into the urinary tract, provided 
that the other clinical signs correspond with this assumption. 
The occurrence of thread-like formations is very characteristic 
of gonorrhea and gonorrheal sediment. They consist of pus 
cells glued together with mucus. 

In the females the pus sediment may come from the vagina. 
To exclude such a possibility, either the vagina must be thor- 
oughly irrigated before the urine is voided or the urine must 
be drawn with a catheter. 

With alkaline fermentation a pus sediment is oftentimes 
converted into a ropy, gelatinous mass by the swelling of the 
pus corpuscles. 

Microscopic Examination. — The pus corpuscles voided in 
the urine vary in their microscopic appearance, which depends 
partly upon the length of time since their escape from the 
blood-current, and partly upon the consistence of the urine, 
or upon the nature of the affection in question. Sometimes they 



TUBE CASTS. 353 

are very cloudy and shrunken, so that the nuclei can be seen 
only after the addition of acetic acid (usually polynuclear or 
with nuclei irregularly crumpled) ; sometimes in an alkaline 
urine they are much swollen and glossy, and in this case also 
the nuclei are not easily seen. 

Donne's Test for Purulent Sediment. — To the suspected 
sediment add a small piece of caustic potash, and stir with a 
glass rod. Pus will become thick, tough, and gelatinous, while 
mucus will become flaky and thin. 

SiGNiFiCAi^CE OF Teue Pyuria. — The presence of pus in 
the urine indicates the presence of an inflammatory process in 
the genito-urinary tract the location of which can, in some 
measure, be determined by the character of the associated epi- 
thelial cells. 

EPITHELIA. 

The epithelial cells found in urine may come from any part 
of the genito-urinary tract. Their forms vary greatly, and 
with a knowledge of the characteristic cells belonging to the 
different regions, it is possible, with more or less certainty, to 
determine their origin by, their appearance. The pus cell may 
be taken as the standard of size. 

Cells from the tubules of the kidney are round and about 
one-third larger than a pus cell. 

From the pelvis of the kidney, twice the size of a pus cell 
and cuboidal or pear-shaped. 

From the ureter, round and slightly smaller than from the 
pelvis. 

From the bladder, flat and square. These are the largest 
cells encountered, except those from the vagina. 

From the urethra, smaller than from the bladder ; they may 
be cuboidal or columnar. All epithelial cells are gTanular ana 
contain a relatively small nucleus. 

TUBE CASTS. 

General Considerations. — In the presence of albimiinuria or 
hematuria, microscopic examination of the urinary sediment will, 
as a rule, reveal tlie presence of tube casts. Occasionally some 



354 THE URINE. 

varieties of casts will be found in urine which show neither 
albumin or blood. 

The Sediment. — In order to obtain a sediment for micro- 
scopiq examination, some method of precipitating and of con- 
centrating this precipitate is necessary for a conclusive exami- 



FiG. 65.— "Large White Kid- 
key." X350. 

Hyaline cast. g. Spiral 
cast, w. Waxy cast, f. Fat- 
granule cast with : n, fat 
needles. Still finer needles of 
this type upon the neighboring 
fat-granule spherule, fc, Fat- 
granule cell, I, Leukocyte, s. 
Vaginal epithelium. t, Fat- 
droplets. 




Fig. 66.— Chronic Bright's 
DISEASE (Chronic Paren- 
chymatous AND Inter- 
stitial Nephritis). 
X 350. 

7^, g, e, w, Hyaline, granular, 
epithelial, and waxy casts, ep, 
Renal epithelium, vep, Quite 
uniformly fatty renal epi- 
thelium. 



nation. A centrifuge is the most rapid as well as the safest 
method to emplo3\ This< process only requires from 15 to 20 
cubic centimeters of urine, and can be accomplished in a few 
minutes. After centrifugation a part of the supernatant urine 
is poured. off and then, with the aid of a pipette with a small 
point, a drop or two ia drawn up and transferred to a clean 
microscope slide for examination. 



TUBE CASTS. 355 

The method of employing a conical glass may be used if a 
centrifuge is not at hand. Sedimentation by this means re- 
quires a number of hours, and, if care is not taken to prevent 
bacterial contamination and if the specimen is not kept in a cool 
place, the specimen may decompose and the morphologic ele- 
ments be destroyed. 

The sedimentation glass is valuable for roughly estimating 
the amount of gross sedimentation in phosphaturia, pyuria, etc. 
(Fig. 67). 

Varieties of Casts. — Hyaline casts are almost transparent 
and appear as ground glass. They have a delicate but definite 
outline, and are quite friable. 




ABC 

Fig. 67.— Sedimentation Glasses. 

A, Clear. B, Slightly cloudy with beginning precipitation. 

C, Sedimentation complete. 

Strong illumination of the field may obscure them entirely, 
as they are very delicate in outline and structure. They are best 
seen with the plane side of the reflector, and side illumination 
which should not be too bright. 

The particular significance of hyaline casts is not yet posi- 
tively settled. They occur in advanced grades of nephritis and 
again in transient albuminurias, and even in the absence of 
demonstrable albumin; they are frequently seen during the 
course of fevers, particularly typhoid fever. 

Granular Casts. — These are more opaque than the hya- 
line, and are, therefore, more easily seen. The granules are 



356 THE URINE. 

numerous^ and upon close examination will be found to permeate 
the matrix of which the cast is partly composed. This will serve 
to differentiate them from the psendo-grannlar casts^ which are 
merely hyaline casts to which granular debris has become at- 
tached during centrifugation or sedimentation. These latter 
are of no more significance than hyaline casts. Granular casts 
are probably composed of a hyaline matrix which has undergone 
degeneration. They frequently show fragmented cells and fat 
globules in their structure. The continued occurrence of hya- 
line and granular casts in the presence of a permanent albu- 
minuria, usually denotes chronic interstitial nephritis. 

Hyalo-granular Casts. — Hyaline casts are occasionally 
found, parts of which are distinctly granular throughout their 
substance. They apparently represent a stage of cast-formation 
between the hyaline and granular vajieties, and their significance 
is probably the same as hyaline casts. 

Epithelial Casts. — These casts are composed of renal 
epithelial cells (slightly larger than pus cells), grouped in the 
form of a short cylinder and cemented together with a hyaline 
or mucoid matrix. The cells may be either whole or fragmented, 
clear, opaque, or granular. They are significant of an acute 
desquamative process, resulting from renal inflammation. 

Fatty Casts. — These casts may possess any of the charac- 
teristics of the preceding varieties^ and present in addition free 
fat-globules scattered throughout the cast. They are considered 
as proof of fatty degeneration of the kidney. 

Blood Casts. — These casts are composed either of coagu- 
latedi blood, in which innumerable corpuscles in various stages 
of disintegration are embedded, or they may represent a hyaline 
matrix in which appears a varying number of red blood-cells. 
Some hyaline casts, which show a few blood-cells adherent upon 
their surfaces, may be simply hyaline or granular, to which the 
red cells have become attached in the bladder or after voiding. 
The presence of true blood easts indicates hematuria of renal 
origin. 

Bacterial Casts. — These resemble the granular variety 
except that they are more closely and more uniformly granular. 
The bacteria may be so numerous that the cast is almost opaque. 



CYLINDROIDS. 357 

They denote the occurrence of an acute bacterial infection of 
the kidne}^, and are rarely found. 

Waxy Casts. — These are of rare occurrence, and are prob- 
ably simply a dense variety of the hyaline cast. 

Crystalline Casts. — As the name implies, they are crys- 
talline in nature, being composed usually of uric acid, and more 
rarely of oxalates. They are very rarely encountered. 

CYLINDROIDS. 

These are narrow, ribbon-like bands which usually present 
longitudinal striations, which may or may not extend through- 
out the entire length of the cylindroid. 

They are essentially hyaline in nature, and are probably 
formed from a hyaline basis. They are of renal origin, and are 
encountered in about 75 per cent, of cases which ordinarily come 
to the physician in the course of practice. 

Their significance is slight, though they are considered to 
indicate a degree of kidney irritation. In this connection it is 
of interest to note that when found they are many times accom- 
panied by a high specific gravity, and oxaluria, with or without 
the presence of indican. 

Microscopic Appearance. — Cylindroids appear as faint, 
ribbon-like bands, seen best by low illumination, and frequently 
exceeding in length the diameter of the field of the microscope 
when viewed through an objective of moderate power. Longi- 
tudinal striations may usually be detected, often running 
through only a portion of their length, the ends of which, after 
making one or more graceful curves, terminate in a gradual 
taper. 

Cylindruria with Albuminiiria. — This condition may occur 
as a transitory phenomenon after such violent exercises as bicycle 
racing, foot-ball or rowing; also in chronic constipation or 
after the ingestion of moderate or large amounts of alcohol, par- 
ticularly by those unused to it. Continued ingestion of the 
salicylates has been found to produce it. It is often present 
during attacks of intestinal indigestion associated with oxaluria. 



358 THE URINE. 

THE QUANTITATIVE DETERMINATION OF CYLIN- 
DROIDS AND HYALINE CASTS, WITH SPECIAL 
REFERENCE TO LIFE-INSURANCE EXAMINATIONS. 

It often becomes necessary to record the relative number of 
casts in a given specimen, especially in reports of life-insurance 
examinations by examiners in the field, whose records must be 
interpreted by the medical director in the home office. 

For this rough quantitative determining a fresh specimen is 
shaken to prevent sedimentation; then exactly 15 cubic centi- 
meters are centrifuged at the usual rate for exactly three 
minutes. From the sediment a large smear is made and covered 
with a cover-glass. 

Significance. — According to studies by Wm. Muhlberg, who 
suggested this technical! from the standpoint of large insurance 
companies, the following valuation can be given to the findings. 
One or two hyaline casts are not considered of any significance, 
provided the urine has not a low specific gravity. Three to 5 
hyaline casts call for another specimen for examination, and if 
the same number of casts is again found, the risk is rejected. 
More than 5 hyaline casts in a single specimen of 15 cubic centi- 
meters of urine calls for rejection of the risk. 

Epithelia. — Various forms of epithelia are found in 
nearly all specimens of urine, and careful note should be made 
of their form and size, as well as of the condition of their pro- 
toplasm and nuclei. They are highly refractive, and are there- 
fore plainly visible under the microscope, their outlines stand- 
ing out clear and distinct. The general character of the 
epithelia found corresponds to the usual three varieties found 
throughout the body, viz. : squamous or flat, cuboidal, and 
columnar or cylindrical. All epithelia are more or less granular 
and possess one or more nuclei, though the latter are not always 
visible, having been disintegrated or become obscured by granu- 
lar degeneration of the protoplasm. Epithelia are subject to 
certain physical alterations when in the urine. By absorption 
of water they swell up and become more regular in outline, the 
small forms thus becoming spherical. The small cuboidal or 
columnar epithelia are most important, since they probably 



11 Medical Record, vol. Ixxxviii, No. 26. 



CYLINDROIDS AND HYALINE CASTS. 359 

come from the tubules of the kidney. The larger varieties of 
columnar and squamous cells come from the lower portion of 
the genito-urinary tract, the largest cells of all being the 
squamous vaginal epithelia. 

Heart-failure Cells in the Urine. — Epithelial cells con- 
taining brown pigment granules occurring in the sputum have 
long been considered of diagnostic value, their appearance, as- 
sociated with the chronic passive congestion of heart disease, 
having led to their being commonly known as "heart-failure 
cells." Similar pigment granules have been noted in the kidney 
epithelia during congestion. Pathologists who have studied them 
state that the pigment is of two types: (a) large dark-brown 
or black masses found in the neighborhood of hemorrhages, and 
(h) light-yellow to brownish-yellow granules occurring in the 
epithelium of the tubules without association with gross hemor- 
rhage. 12 These cells have been shown to be similar to those found 
in the lungs in that they are of epithelial origin, but to differ 
from the latter in that the granules are rarely composed of 
hemosiderin. While the presence of such granules in the kidneys 
has been up to the present of little more than academic interest, 
an article by Bittorfi^ holds out the hope that they may prove 
to be of actual clinical and diagnostic importance. This hope 
is based upon this author having found such cells fairly con- 
stantly in the urine in cases of passive congestion of the kidneys 
as a result of heart disease. The cells found ini the urine are of 
the type containing light-yellow granules. They are seen as 
more or less swollen, polymorphous, but usually polygonal cells 
of various, often large sizes, with indistinct, large, round nuclei. 
There may be several together or they may be attached to casts. 

Significance. — Their occurrence in the urine associated with 
casts and a few scattered red blood-cells is an objective evidence 
of a circulatory affection of the kidneys, especially if the common 
sources of kidney hemorrhage, as tumors, infarcts, and acute 
nephritis, can be eliminated. 

Pus AND Blood Cells. — For the satisfactory study of these 
small elements, a higher power objective than that used for the 
study of crystals and epithelia is required. Oblique light, with 

12 Review, Medical Record, October 23, 1909. 

13 Miinch. med. Wochen., August 31, 1909. 



360 THE URINE. 

moderate illumination, will be found best. Occasionally great 
difficulty is experienced in differentiating blood, pus, and small, 
round epithelial cells. This confusion is not so likely to occur 
when all varieties are present in sufficient quantity in one speci- 
men to admit of careful comparison; but when only a few 
scattered cells of one variety are found, mistaken identity is 
quite possible. It is well, as a general guide, to bear in mind 
the relative size of these different elements. Pus-corpuscles are 
the most common, and these should be thought of first. They 
are identified as small granular discs, having multi-nuclei. 
Somewhat smaller in size (about one-third) will be noted the 
pale, non-granular, non-nucleated discs which are the red blood- 
cells. While at least one-third larger than the pus-cells, the so- 
called renal epithelia present a granular protoplasm with large 
nuclei. 

Finally, after determining the individual elements in a 
given specimen, it is important to note the degree of degenera- 
tion and the presence of fatty change, particularly occurring in 
the casts; and, lastly, the number of each formed element per 
field or per drop should be determined, this observation 
being important in following the course of any pathologic con- 
dition, as it is similar to the repeated quantitative determina- 
tion of albumin or sugar. 

Bacteriuria. — The presence of excessive numbers of micro- 
organisms, their motility, and even some of their morphologic 
characteristics may be determined during the microscopic 
examination of the sediment; a complete and satisfactory 
examination of their detail and identity cannot, however, be 
determined except by special bacteriologic technic, for which the 
reader is referred to larger works on bacteriology or urinary 
diagnosis. 

Spermaturia. — ^The presence of semen in the urine will give 
positive reactions for albumin. It may be present in the urine 
after coitus, after nocturnal emissions, or in spermatorrhea. 

Microscopic examination will reveal the characteristic ele- 
ments which are motionless, but which resist decomposition for 
days. 

Chyluria. — Under very occasional circumstances the urine 
may contain chyle, which gives it the appearance of milk, and 



THE INORGANIC SEDIMENT. 3^1 

forms on standing a cream-like layer at the top. It responds to 
all the tests for albumin. 

Microscopically, much fat is found in the form of innumer- 
able highly refractive globules, which are soluble in ether. 

A large amount of fat in the urine almost always signifies 
chyluria (lipuria). The urine is albuminous, of a milk-white 
to a cloudy-yellow appearance, sometimes even slightly blood- 
tinged, neutral or faintly acid, forms a cream-like layer, and 
often contains small coagula. The latter may form within the 
body as well as after the urine is voided. A microscopic ex- 
amination shows that the fat is subdivided much more freely 
in the urine than it is in milk. No distinct fat-drops can be 
seen, but extremely finely divided, almost invisible, fat granules. 
These granules furnish the cloudiness and cream-like layers. 
By shaking a little of this cloudy urine with ether the fat may 
easily be removed, and demonstrated by evaporation of the 
ether residue. 

THE INORGANIC SEDIMENT. 

To obtain a specimen of inorganic sediment, it is preferable 
to centrifuge the freshly voided urine rather than to wait for 
the slower process of sedimentation. The use of the centrifuge 
precludes the possibility of the process of decomposition so 
altering the specimen that the original picture is destroyed. 
Alteration in reaction will so change the character of the crys- 
talline deposit that it will be useless from a diagnostic stand- 
point. A decomposed alkaline urine will often present such a 
mass of phosphatic sediment that the less voluminous, but more 
important, elements are greatly obscured and may be over- 
looked. 

Preparation of the Slide. — The sediment should be taken 
from the bottom of the centrifuge tube (Fig. 68) by means of a 
small pipette with a long, slender tip. Not more than a drop or 
two should be allowed to enter the tube. This is particularly 
important when the amount of sediment is small. If the sedi- 
ment is large and dense it should be diluted with a drop or two of 
distilled water and a cover-glass placed upon it. Much time will 
be saved by first examining the preparation with the % or % 
objective, which is convenient for locating an interesting part 



362 THE URINE. 

of the slide, upoii which the high-power objective may be focused 

for more careful study. 

Crystalline Deposits. (See Plates IX, X, and XI.) 

Acid Group. — Uric Acid (Plates IX and XI) : These 

crystals are yellow, reddish-brown or brown in color. The most 

characteristic forms are rhombic prisms or lozenge-shaped crys- 



A B C 

Fig. 68.— Centrifuge Tubes. 
A. Correct form of graduated percentage tube (note the slender taper which 
facilitates reading of small per cent.). B, Improper form of graduated tube 
accurate reading of small percentages diflBcult. C, Plain centrifuge tube. 

tals (Plate IX, Figs, a and h). These occur singly, but more 
often they are united in irregular masses. (Other more rare 
forms 'are shown in Plate IX, Figs, c^ d, e, and /; Plate XI, a 
and h.) 

Urates. — The urates, chiefly the urate of sodium and the 
urate of potassium, if they do not appear as an amorphous de- 



PLATE IX. 




a and b. Usual Forms of Uric Acid Crystals. ^, ^, e and f. Less 
Common Uric Acid Crystals. 



PLATE X. 




a and b. Calcium Oxalate Crystals. c, d, e and f. Phosphates. 



INORGANIC SEDIMENT. 363 

posit^ appear as crystals in the forms of needles or dumb-bells, 
of reddish-brown color, and also in globular masses which are 
dark-brown and almost opaque, with or without projecting 
spines. 

Oxalates. — The usual form of calcium oxalate in the urine 
is a perfect octahedron without color. More rarely they appear 
in the conventional hour-glass form (Plate X, a and h). This 
form is somewhat similar to the urate from which it may be 
distinguished by the total absence of color in the oxalates. 

Carbonates. — These are rare, but when present evolve bub- 
bles of gas when treated with hydrochloric acid under the micro- 
scope. 

Sulphates. — This is a rare form of deposit which, when 
present, appears as fine, feathery crystals. Frequently a number 
of crystals radiate from a common center. 

Alkaline Group. — Phosphates: These may occur as a 
semi-opaque amorphous deposit without color. More commonly 
they appear as the characteristic coffin-lid crystals. A less com- 
mon form of crystalline phosphatic deposit (Plate X, c, d, e, 
and /) appears as fine, branching, feathery crystals, which have 
been likened to the needles and branches of the pine tree. 

Ammonium Urate (see Pig. 60). — These are characteristic 
of the uric acid and urate group in that they are yellow or 
brownish in color. In alkaline urine the urates appear as fine, 
feathery spheres of varying size, resembling to some extent 
chestnut burrs. 

Cholesterin. — This is a rare form of deposit which appears 
in the form of irregular, flat platelets whose sides follow the 
characteristic lines of a parallelogram, the angles of which are 
often irregular. Not infrequently the platelets occur in over- 
lapping groups. (Plate XI, c.) 

Cystin. — This is a rare deposit. When present it appears 
as irregular transparent plates of varying size, often in over- 
lapping groups. (Plate XI, d.) 



364 THE URINE. 

METHOD TO DETERMINE ROUGHLY THE NATURE OF 
COMMON UNORGANIZED URINARY DEPOSIT. 

1. Warm the deposit and some urine. If it dissolves, the 
deposit is composed of urates. If it is unaffected by heat, the 
deposit is either phosphates, uric acid, or calcium oxalate. 

2. Warm a fresh portion of urine with acetic acid. If the 
sediment dissolves, it is composed of phosphates; if it does not 
dissolve, it is uric acid or calcium oxalate. 

3. Add to this undissolved portion some HCl and heat 
again. If the sediment now dissolves, it is calcium oxalate; if 
it does not dissolve, it is composed of uric acid. 

URINARY CONCRETIONS. 

General Considerations. — About 75 per cent, of all urinary 
concretions are either uric acid or urates. Next in frequency 
are found the calcium oxalate or mulberry concretions. More 
rare primary concretions may consist of blood, cystin, xanthin, 
calcium phosphate, or calcium carbonate. 

Secondarily, any one of these formations may become cov- 
ered with a whitish layer of mixed phosphates. These arc pre- 
cipitated upon the original concretion by ammoniacal fermenta- 
tion, which occurs in the bladder, secondary to the presence of 
the calculus. 

Analysis of the Concretion. — 1. Burn a portion upon a 
piece of platinum foil in a Bunsen flame or blow-pipe. 

A. If it chars greatly and leaves little ash, it may be uric 
acid, urates, cystin, xanthin or blood. 

1. If it gives the murexid test, it is uric acid or urates. 

2. If it dissolves in boiling water, it is urates. 

3. If it does not dissolve in boiling water, it is uric acid. 

4. Cystin and xanthin are very rare forms of concre- 

tions which require special tests for their detection. 

B. If it chars very slightly and leaves considerable ash it 
may be phosphates, oxalates or calcium carbonate. 

1. Treat a fresh portion with dilute HCl. 

(a) If it dissolves with effervescence, carbonates are 
present. 



THE DIAZO REACTION. 365 

(b) If it is soluble without effervescence, phos- 
phates or oxalates are present. 
(See C, 3, below.) 
3. Treat a portion with dilute acetic acid. 

(a) If it dissolves with the aid of heat, it is phos- 
phatic. 
C. 1. If it fuses to a bead oni platinum foil, it is urates. 

2. If it does not fuse, it is calcium phosphate. 

3. If it is insoluble in either of the above acids, it is 

calcium oxalate. 

THE DIAZO REACTION. 

The Reaction. — The ammoniacal solution of the anhydride 
of para-diazo-benzin-sulphonic acid has the property of devel- 
oping a salmon pink or red color reaction with certain patho- 
logic urines. Normal urine never gives this reaction. 

The Test (for preparations' of solutions see Appendix). — 
Take 5 cubic centimeters of solution "A" and add three drops 
of solution "B,^^ and mix together in a clean test-tube. To this 
add 5 cubic centimeters of urine and again mix. Now allow a 
few drops of ammonia water to flow down the side of the tube. 
As it comes in contact with the mixture in the tube a pinkish 
or red color indicates a positive reaction. Upon shaking the 
tube the entire mixture becomes colored, and the color is also 
imparted to the foam. This reaction does not always appear 
immediately, so a few minutes should be allowed for its devel- 
opment before reporting a negative reaction. 

Feri's Improved Diazo-reaction.i^ — Feri's modification of the 
test consists in the use of a single reagent, marketed under the 
name of "asophorroth P. N.," instead of the fresh mixture of 
sulphanilic acid and sodium nitrite, as recommended by Ehrlich. 
The method is simpler and the reagent permanent. A trace of 
the former is shaken up with a little water in a test-tube and a 
few drops of the urine, made alkaline with sodium hydrate, are 
added. A brilliant red color marks the positive reaction. 

Significance of the Beaction. — It is not yet definitely known 
what particular substance or substances in the urine yield this 



i4Wien. klin. Woclien., No. 24. 1912. 



366 THE URINE. 

reaction. It may occur in the presence of several aldehydes, 
ketones, and phenols. 

A positive reaction is usually obtained with the urine of 
typhoid fever patients after the fourth day of the disease. Its 
continued absence, however, does not preclude the possibility of 
this disease. It is also positive in some cases of measles, pul- 
monary tuberculosis, etc. 

Eusso's Test (Methylene-blue Eeaction). — This test 
has been recently advanced and seems to have somewhat more 
diagnostic importance than has the diazo-reaction. The technic 
is very simple and is as follows : 4 drops of a 1 to 1000 aqueous 
solution of methylene-blue are added to 4 or 5 cubic centimeters 
of suspected urine. If the reaction be positive the mixture turns 
to an emerald or mint-green hue. A light-green or bluish- 
green tint shows a negative reaction. The positive reaction is not 
affected by boiling the urine or by the previous ingestion of such 
compounds as phenacetin, salol, quinin, and calomel. The diffi- 
culty in the application of the test comes in the ability to 
recognize the various tints of green which may be present. With 
a little practice, however, a positive reaction may be readily 
detected, especially if a control test be made with normal urine. 

This test is shown as early as the second day of typhoid 
fever and persists throughout its course. The mint-green hue 
is first observed, the emerald-green tint appearing as the disease 
progresses. If the course is favorable the color tone becomes 
more and more bluish, while if unfavorable the emerald tint per- 
sists. This test is also positive in measles, small-pox, chronic and 
suppurative tuberculosis, but it is negative in varioloid, varicella, 
scarlet fever, miliary tuberculosis, appendicitis, and malaria. 

This test is as simple and apparently just as reliable as is 
the diazo-reaction, being especially valuable in differentiating a 
typhoid from a miliary tuberculosis. 

Fallacy. — Urines containing bilirubin react to this test, so 
that this fallacy must be borne in mind. (See page 349.) 



EXAMINATION OF THE URINE. S67 

EXAMINATION OF URINE FOR SUBSTANCES INTRO- 
DUCED INTO THE BODY FROM WITHOUT. 

(Drugs and Poisons.) 

Detection of Lead. — A bright strip of magnesium, free from 
lead, is placed in the urine and left there for some time; the 
deposit which forms is dissolved in nitric acid, and then tested 
according to the methods of inorganic analysis. ^^ 

If the urine contains but a small amount of lead this method 
will not give the desired result; then a larger quantity of urine 
must be used, the organic substances destroyed with HCl and 
potassium chlorate, and the lead sought for in the evaporated 
residue. 

Detection of Mercury. — To 500 to 1000 cubic centimeters of 
urine, add 2 to 4 cubic centimeters of HCl; then digest at 60° 
to 80° C, for five or ten minutes in a flask with a few bright 
strips of brass or copper. The metal is then washed with 
water, then with alcohol, and finally with ether, and dropped 
into an ignition tube (glass tube of high-fusing point). This is 
then brought to a red heat, care being exercised to keep the 
upper end of the tube cool. The mercury which has become 
amalgamated with the metal becomes volatilized, and is rede- 
posited in the upper cold end of the tube, where it is seen as a 
bright mirror, or, if a small amount, the individual globules of 
mercury may be seen by a hand-lens or the low power of the 
microscope. 

Detection of lodin. — lodin occurs in the urine as potas- 
sium iodid after the internal or external application of iodin 
or one of its combinations. It may easily be demonstrated as 
follows: A few cubic centimeters of urine are boiled with a 
piece of starch of about the size of a pea until the latter is dis- 
solved. After cooling, the fluid is carefully overlaid with con- 
centrated nitric acid. If iodin is present a blue-violet ring, 
which gradually disappears, is formed at the line of junction of 
the two fluids. A second method is to add to the urine a few 
drops of crude nitric acid and a less number of drops of chloro- 
form, and then shake gently. If iodin is present the 
chloroform, which sinks to the bottom of the tube, will be 



15 See Marshall's "Medicus," J. B. Lippiucott Co. 



368 THE URINE. 

colored rose-red or violet. Both the above tests are very 
delicate, but if the urine contains only a small trace of iodin, 
the chloroform test is not very conclusive, since the nitric acid 
may set free indol and skatol pigments, as well as urorosein, 
any of which will color the chloroform in a similar manner. 
But in this case the urine itself appears more deeply colored 
than the chloroform. 

Detection of Bromin. — The test for bromin is performed 
in exactly the same manner as iodin test, except that a few 
drops of a calcium chlorid solution and hydrochloric acid are 
used to liberate the bromin. If bromin is present the chloro- 
form will be colored yellowish-brown. This test, though far less 
delicate than the iodin- test, is sufficiently accurate to recognize 
the therapeutic ingestion of large doses of bromin salts, and is 
chiefly of interest in verifying the diagnosis of suspected bro- 
mism. 

This method may be uncertain because of the discoloration 
of the chloroform by urinary derivatives. To prevent this source 
of error 16 cubic centimeters of urine, to which have been added 
2 cubic centimeters of caustic potash and 2 cubic centimeters 
of potassium nitrate, are evaporated and incinerated, the ash 
dissolved in water, and the resulting solution tested for the 
presence of bromin, as above. 

Detection of Salicylic Acid. — Dilute ferric chlorid is added 
to the urine drop by drop. If the latter becomes a more or less 
intense violet, the reaction is positive. Salicylic acid and its 
salts, in which latter form the administered salicylic acid partly 
appears in the urine, both give this reaction. 

Detection of Phenol. — Phenol appears in the urine largely 
as phenol sulphate. Ferric chlorid will produce a violet-blue 
color in the distillate from phenol urine, to which 5 per cent, 
sulphuric acid has been added. Phenol urine turns dark or black 
on exposure to the air, when sufficient time is allowed for oxida- 
tion to occur. 

Detection of Antipyrin. — ^The urine appears dark and is 
dichroic, i.e., in reflected light, greenish; by transmitted light, 
reddish. A permanent brown-red color gradually appears upon 
the addition of ferric chlorid solution. 



TESTS FOR FUNCTIONAL RENAL CAPACITY. 369 

Detection of Phenacetin ( Acetphenetidin) . — The urine is 
dark yellow and turns reddish-brown on the addition of ferric 
chlorid solution. The color gradually becomes black after pro- 
longed standing. 

Detection of Antifebrin. — The urine is extracted with chlo- 
roform, and to the extract mercuric nitrate is added. The mix- 
ture is then heated^ and if a green color is produced antifebrin 
is present.!^ 

Detection of Pyramidon. — ^The urine is frequently clear 
purplish-red in color, and deposits a sediment composed of 
small, red needles. If the urine is mixed with an equal volume 
of a 2 per cent, solution of ferric chlorid, a dark-brown, ame- 
thyst color is produced. 

Detection of Tannin. — Tannin is eliminated in the urine 
partly as gallic acid. Urine which contains tannic and gallic 
acids turn a deep blue-black upon the addition of ferric chlo- 
rid solution. 

Detection of Balsam of Copaiba and Sandalwood Oil. — After 
the administration of copaiba the urine will reduce cupric oxid 
(Trommer^s test), but not bismuth (Nylander^s test). If HCl 
is added to the urine drop by drop, a precipitate of resinous 
acids appears with a reddish or violet coloration. Also after the 
administration of sandalwood oil the urine has reducing prop- 
erties, and exliibits a precipitate of resinous acid when HCl is 
added, but it will be of a reddish-brown color. 

Detection of Santonin. — Santonin urine is of a saffron-yel- 
low or greenish color. The addition of sodium hydrate will 
turn it a rose-red. If this rose pigment is shaken with amylic 
alcohol, it will be immediately taken up by the latter, giving it 
an intense and beautiful color, while at the same time the urine 
is decolorized. 

TESTS FOR FUNCTIONAL RENAL CAPACITY. 

Since the original work of Geraghty and Rowntree,^"^ 
who first demonstrated the practicability of the phenolsulphone- 
phthalein test, in the study of renal efficiency, subsequent studies, 
both by these observers and by many other clinical investigators, 

16 Yvon : Jour, de pharm. et de cheniie, No. 1, 1SS7. 

17 Jour. Pharm. and Exp. Therap., July/ 1910, i. 579. 

24 



370 THE URINE. 

have repeatedly verified their results. This test has now gen- 
erally superseded the earlier colorimetric tests^, such as the indigo- 
carmin, the rosanalin, etc., because of its uniform reliability, 
and especially because the technic is one well suited to routine 
clinical work, possessing no inherent or technical difficulties, 
eliminating all difficult or pain-producing procedures, and being 
easy of interpretation. For these reasons, the author inten- 
tionally omits other tests of a similar nature, referring the 
reader desiring more extensive information to larger works on 
diagnosis or urology. 

The various steps of the investigation, as originally laid 
down by Geraghty and Eowntree, remain unchanged except for 
slight and unimportant modifications, the more valuable of which 
will be referred to below. 

The test depends upon the fact that phenolsulphonephtha- 
lein when introduced into the body is excreted with extraordinary 
rapidity and appears in the urine normally within a few minutes 
after injection, while in alkaline solution a brilliant red color 
is produced which is ideally adapted for quantitative colorimetric 
estimations. 

Technic of Geraghty and Eowntree. — Direct the patient to 
take 300 to 400 cubic centimeters of water from twenty minutes 
to a half-hour before administering the drug. This insures free 
urinary secretion and prevents delayed time of appearance due 
to lack of secretion. The employment of a catheter to facilitate 
collection of the urine, as at first advocated, has largely been 
discarded, because of the uniformity in the time of the appear- 
ance of the drug after its administration. This has been found 
to be fifteen minutes, so that, in all examinations, fifteen minutes 
are allowed to pass before counting the first hour of secretion. 
Neither is it necessary to commence with an empty bladder, as 
the quantitative estimation depends solely on the quantity of the 
drug excreted, and not on the volume of urine, which may be 
50 or 500 cubic centimeters without affecting the result. 

Only in cases of urinary obstruction and for estimating 
separately the function of each kidney will catheterization have 
to be resorted to. 

One cubic centimeter of a solution containing 6 milligrams 
of the drug is injected intramuscularly (it may be given sub- 



TESTS FOR FUNCTIONAL RENAL CAPACITY. 3 71 

cutaneously or intravenously, if desired). The urine is first 
collected at the end of one hour and fifteen minutes, again an 
hour later, and it is usually advisable to collect a third specimen 
at the end of the next hour. 

Sufficient NaOH (10 per cent, solution) is added to the 




Fig. 69.— Ddboscq Colorimeter, 

collected urine to make it decidedly alkaline, as the character- 
istic pink reaction is only developed in a definitely alkaline 
solution. The collected solution is placed in a 1-liter measuring 
flask and diluted with distilled water to the 1-liter mark. This 
solution is thoroughly mixed and then a small filtered portion 
used to compare with the standard. 

The Colorimeter. — ^In the above as in many of the newer 
clinical methods used in blood and urinary analysis, an instru- 



372 



THE URINE. 



ment known as a colorimeter is emplo3'ed. With it we are able 
to accurately measure and compare the respective depths of 
color in two solutions and thus are enabled to calculate the 
comparative (see page 313) amounts and substances which form 
compounds in a quantitative manner. The most accurate 
(within 1 per cent.) of these instruments is the Duboscq col- 
orimeter (see Fig. 69). This enables one to compare two col- 
ored solutions in the same optical field. A recent improvement in 
this instrument substitutes cylinders for prisms, and is to be 




Fig. 70.— Hellige or Universal Colorimeter. 



preferred since it renders the instruments available for the com- 
parison of cloudy solutions and bacterial suspensions. When 
employed for this purpose the instruments act as a nephelom- 
eter. This latter type of instrument when working as a colorim- 
eter is arranged to accurately vary the depth of the color solu- 
tions employed, through which the light passes; this layer of 
fluid being accurately indicated in millimeters on a vernier 
scale located at the back of the instrument. The final reading 
is made directly by means of comparison between the position 
of the two cups at the moment when the two halves of the 
optical field are of the same shade and density of color. 



TESTS FOR FUNCTIONAL RENAL CAPACITY. 373 

There are many varieties of colorimeter which may be used 
in place of the Dnboscq. These are less expensive and usually 
less accurate, bnt not enough so to condemn them. The Hellige 
(see Fig. 70) has found favor at the hands of many clinical 
laboratory workers and can be recommended by the author. It 
is advisable to make a comparison soon after voiding, and, if 
this is not possible, to render the urine distinctly acid, as the 
presence of alkali causes the red color to gradually fade in a 
few hours. 

Additional Teciinic. — In the presence of marked urinary 
obstruction, or retention, a catheter should be introduced and 




Fig. 71.— Dunning Colorimeter. 

allowed to remain until the end of the third period of collection, 
three hours and fifteen minutes. To investigate the excretory 
power of each kidney separately, the catheterizing cystoscope 
will have to be employed, and the urine from each kidney col- 
lected and examined separately. 

In instances in which the pigment elimination is so slight 
as to be practically lost by the usual dilution up to 1 liter, the 
voided or catheterized specimen, after being alkalinized with 
20 cubic centimeters of a 10 per cent, sodium hydrate solution, 
which is necessary in every instance, in order to bring out the 
color value, the dilution should be made only to 250 or 500 
cubic centimeters and allowance made for the fractional dilution 
when comparing with the colorimeter.is 

• Significance of Findings. — Normally, the largest amount 
is eliminated at the end of the first hour and fifteen minutes, 

18 w. E. Robertson : N. Y. Med. Jour., May 16, 1914. 



374 



THE URINE. 



the amount varying from 30 to 50 per cent., with 15 to 25 
per cent, at the end of the next honr, and merely a trace 
in the third specimen. Abnormally this condition is reversed, 
and the greatest amount is eliminated in the second or even the 
third hour, and in uremia or impending uremia elimination 
is often too slight to permit of definite reading in any of the 
specimens (Robertson). 




Fig. 72.— Kober Colorimeter. 



Following intravenous injections the drug appears in three 
to five minutes; 35 to 45 per cent, is eliminated in the first 
fifteen minutes; 50 to 65 per cent, in the first half-hour and 63 
to 80 per cent, during the first hour. For general use the lumbar 
intramuscular method is advocated especially when edema is 
present (Geraghty and Eowntree). 

Effect of Drugs. — The output does not seem to be in- 
fluenced by the previous administration of the different diure- 
tics, as caffeine, urea, calomel, etc., whereas those affecting the 
blood-pressure and osmotic tension, as the nitrites, and digitalis 
may glightly decrease the phthalein output. 



TESTS FOR FUNCTIONAL RENAL CAPACITY. 375 

Value in Surgery. — ^Tests of 100 cases show it to be a 
valuable guide as to the ability of the patient to stand anesthesia 
during operation. 

Application of test to determine functional activity of in- 
dividual kidneys is useful in cases of unilateral and bilateral dis- 
ease ; here the urethral catheter must be used. Under these con- 
ditions the time of injection is recorded, and also the time of 
appearance of the drug on each side. Starting from the time of 
the appearance, the collection is then continued for one hour. 
The amount of drug in each specimen is then estimated as above. 

Eesults in Normal Cases. — The time of appearance on 
two sides is usually simultaneous. The amount normally ex- 
creted of each kidney will be practically the same. Under 
surgical conditions, the test is of great value in demonstrating 
the doubtful diseased condition of one kidney and to prove 
the functional capacity of the other kidney in cases where opera- 
tion on the kidney is indicated. 

It is of immense value from a diagnostic and prognostic 
standpoint in nephritis, inasmuch as it reveals the degree of func- 
tional derangement in nephritis, whether of the acute or chronic 
variety. 

In the cardiorenal cases the test may prove of value in 
determining to what degree renal insufficiency is responsible for 
the clinical picture presented. 

The test has proven of value not only in diagnosing uremia 
from conditions simulating it, but has also successfully indicated 
that uremia was impending when there was no clinical evidence 
of its existence at the time. 

The test has proven of great value in revealing the true 
renal condition in cases of urinary obstruction. It is here of 
more value than the urinary output of total solids, urea, or total 
nitrogen, and enables the surgeon to select a time for operation 
when the kidneys are in their most favorable functional con- 
dition. The improvement in the renal condition in cases of 
urinary obstruction following the institution of preliminary 
drainage is strikingly indicated by this test. 

To obtain the most accurate results recourse must be had 
to other tests related to renal functional capacity. A complete 
examination should include, besides a repeated phenolsulphone- 



376 THE URINE. 

phthalein test^ an indigo-carmin test, by means of the cysto- 
scope, and an estimation of twenty-four hours' nitrogen 
elimination with the diet under control as far as the nitrogenous 
intake is concerned. " 

URINARY TESTS FOR FUNCTIONAL ACTIVITY 
OF OTHER ORGANS. 

CAMMIDGE REACTION.19 

This reaction is based upon the presence of certain, at 
present unknown, substances, which occur in the urine in pan- 
creatic disease associated with fat necrosis, the detection of 
which is based upon some special reactions in which phenylhy- 
drazin hydrochlorid plays an active part. 

Many observers have followed the methods of Cammidge in 
the determination of this reaction and its relation to disease of 
the pancreas. Cammidge himself, realizing the non-specificity 
of the original A- and B-reactions, later added a third or C- 
reaction, in an effort to render the findings more conclusive. In 
spite of this we are as yet unable to place any great confidence in 
the findings of this test, the results obtained by a number of 
careful aiid competent investigators being so at variance that it 
seems doubtful if this test will ever be established upon a sure 
and practical foundation. 

The dtfi&culty may be due in part to the inherent difficulties 
in the technic itself and to the extreme delicacy of the reaction. 
The slightest variation in the performance of its various steps 
often rendering valueless a large amount of tedious and time- 
consuming work, we must conclude, after a careful analysis of 
the available, clinical and experimental data pertaining to this 
reaction and its relation to the diagnosis of pancreatic disease 
that it is still in an experimental stage, and that many uncertain 
and negative results in undoubted lesions of the pancreas make 
it impossible to place a definite value upon the findings; this 
together with the difficulty with the test itself removes the Cam- 
midge reaction as an available clinical procedure from daily use 
by the clinician. 

19 See "Diseases of Pancreas," Robson and Cammidge, 1907, for further in- 
formation. Also Schumm and Heyler : Munch, med. Wochen., Feb. 14, 1909; 
Jour. A. M. A., Aug. 22, 1908; J. B. Schmidt: Mitteilungen aus den Grenzge- 
bieten der Med. und Chir., Jena, July 24, 1909; Speese and Goodman: N. Y. 
Med. Jour., Aug. 14, 1909; Cam m idge ; Lancet, March 19, 1904; Mayo Robson; 
Lancet, page 773, 1904. 



HIROSE TEST FOR FUNCTION OF LIVER. 377 

DIMETHYLAMINOBENZALDEHYD REACTION FOR 
FUNCTION OF LIVER. 

This test, advanced by Ehrlich, is as follows : Prepare a 2 
per cent, solution of p-dimethylaminobenzaldehyd in equal parts 
of concentrated HCl and water. Add a few drops of this solution 
to 5 cubic centimeters of fresh cold urine and allow to stand 
for a few minutes. A positive reaction is indicated by the 
appearance of a cherry-red color, which may be extracted with 
chloroform. Heating facilitates the reaction, but here normal 
urine may give a slight reddish coloration. In the cold normal 
urine gives a greenish-yellow color. 

Significance. — Apparently this coloration is due to the 
presence of metabolic products derived from blood-pigments 
(urobilinogen compounds). It is to be expected, therefore, that 
this reaction would be distinct in diseases of the liver and bile 
passages, although it is not constant even here. It is also 
observed in tuberculosis, pneumonia, typhoid fever, and malaria. 

From his studies of this test, Allen Eustis^o suggests the 
following additional steps : If urobilinogen is present a scarlet- 
red color appears, which persists on dilution with water. While 
urines in which there is no urobilinogen may give a reddish color 
(due to pyrrhol derivatives) on the addition of the reagent, on 
diluting with water the reddish tinge changes to yellow, while 
those urines giving a positive reaction change to pink, the color 
persisting up to a very high dilution. 

HIROSE TEST FOR FUNCTION OF LIVER. 

This observer 21 recommends the use of galactose to measure 
carbohydrate tolerance because the normal limit of tolerance in 
the fasting state is %o ^^hat of cane sugar (or 20 to 40 grams 
daily). 

The test is applied after fasting, with special reference to 
milk, by giving 24 grams chemically pure galactose, and then 
examining small specimens of urine for this sugar during a 
period of twenty-four hours. 

The test possesses no specific value, but may be found to 
be of value in measuring hepatic insufficiency. 

20 New Orleans Med. and Surg. Jour., December, 1912. 

21 Deut. med. Wochen., July 25, 1912. 



XL 

BODY FLUIDS, EXUDATES, TRANSUDATES, 
AND SECRETIONS. 



CEREBROSPINAL FLUID. 

General Considerations. — Since the introduction of lumbar 
puncture by Quincke, the cerebrospinal fluid has gained so much 
importance from a diagnostic standpoint that much of value 
may be learned from a systematic study of this fluid. There is 
little danger in the performance of lumbar puncture, as the 
spinal cord does not reach to the usual point of puncture and 
the fibers of the Cauda equina are sufficiently movable to escape 
the needle. While few effects are observed in the ordinary run 
of cases, a few have been reported in which symptoms of collapse 
are evident. It should be a rule, therefore, to stop proceedings 
if such symptoms arise and also to keep the patient quiet in 
bed for at least twenty-four hours following the puncture, or 
until the pressure in the cerebrospinal cavities may become 
equalized. 

The examination of the cerebrospinal fluid conveniently 
divides itself into three groups: (a) A study of the 
cerebrospinal pressure; (&) an examination of the physical 
characteristics of the fluid, including its general chemistry; a 
microscopic or C3'tologic study; (c) a study of special more or 
less specific reactions and a bacteriologic examination. The most 
valuable of the clinical tests are those proposed by Nonne and 
Appelt, but modified and improved by Eoss and Jones, i and 
later by J^oguchi and Moore. ^ This test is based upon the fact 
that in inflammatory conditions of the meninges the cerebro- 
spinal fluid contains an increased amount of globulin, while in 
other pathologic conditions of the central nervous system, 



1 British Medical Jour., May 9, 1909. 

3 Jour. Exper. Medicine, vol. ii, p. 604, 1909. 

(378) 



CEREBROSPINAL FLUID. 379 

non-inflammatory in nature, the amount of globulin is present 
in amounts not demonstrable by ordinary chemical methods. 
The original test of Nonne and Appelt devised to demonstrate 
this increased globulin content, and known as the ISTonne "Phase 
1'^ reaction, has given way to the modification of Eoss and Jones 
and the Noguchi tests on account of the greater delicacy and 
ease of interpretation of these latter. 

Physical Characteristics. — Normal cerebrospinal fluid is 
colorless, limpid, and practically free from morphologic ele- 
ments. Its specific gravity ranges between 1002 and 1010. It is 
alkaline in reaction, the degree of alkalinity varying between 
15 and 20. It contains a trace of protein and about 0.1 per 
cent, of glucose. 

Pathologically the fluid may be clear or very cloudy, due to 
the presence of leukocytes, erythrocytes, and endothelial cells. 
This cellular admixture may be so extensive as to give the 
appearance of pure pus. In cases of cerebral hemorrhage from 
the ventricles, hemorrhagic pachymeningitis, or traumatic lesions 
of the spinal cord, enough blood may be present to give the 
appearance of practically pure blood, the color varying from a 
bright red to a brownish or greenish red, depending upon the 
length of time it has been shed and has remained admixed with 
the cerebrospinal fluid. This admixture with blood may lead 
to the spontaneous coagulation of the fluid, which may serve as 
a differentiating point between inflammatory and non-inflamma- 
tory lesions. Thus, in tuberculous meningitis very slight coagu- 
lation may be observed, while in the epidemic cerebrospinal 
meningitis the coagulum may be very firm. 

The Chemical Examination of the cerebrospiDal fluid offers 
some points of clinical value. While the albumin content 
normally is much less than 0.1 per cent., it may vary under 
pathologic conditions to as high as 0.8 per cent. The total 
protein, especially the euglobulin portion, is increased in all 
cases of acute inflammations of the meninges, in hydrocephalus, 
in syphilitic and in parasyphilitic diseases of the cerebrospinal 
tract. Glucose is usually present, but may entirely disappear 
under pathologic influences due to an autolysis which is con- 
trolled by the leul^ocytic ferments, the glucose being converted 
into lactic acid. Cholin is present normally as a mere trace, 



380 BODY FLUIDS, EXUDATES, TRANSUDATES, SECRETIONS. 

while pathologically the amount may vary between 0.021 and 
0.046 per cent. 

To Obtain the Specimen.^ — It is very important when with- 
drawing the cerebrospinal fluid to see that too much is not ab- 
stracted at one time. Death has resulted from too rapid and 
too great removal of fluid. This risk need hardly be considered 
in obtaining fluid for purposes of examination, as not more than 
8 to 10 cubic centimeters are required. Untoward symptoms 
are less likely to follow spinal puncture if the patient is kept 
in bed for a few hours succeeding the procedure. 

The control of the needle is important. It is quite possible 
with an 8-centimeter needle to thrust into the peritoneal cavity 
or between the bodies of the vertebra, and by this latter path to 
pierce even the vena cava. Aspiration to start the flow is not 
permissible, as with proper precautions and careful technic a 
dry tap is very rare. 

The Pressure. — For exact pressure-determinations elaborate 
apparatus is not required. For ordinary bedside work the ap- 
paratus about to be described is sufficient: As soon as the sub- 
arachnoid space has been entered, as shown by the appearance 
of fluid in the needle, a thick-walled glass tube of fine bore 
(about 1% millimeters diameter) is joined to the needle by a 
short rubber connection, and the fluid allowed to find its level 
in this tube held verticall}^, and the pressure measured in terms 
of millimeters according to the height to which the fluid rises. 
The capillary error, variations due to the differing specific gravity 
in the fluid, and the change due to loss of fluid from the spinal 
canal, may be neglected for all practical purposes. Under ordi- 
nary bedside conditions, with this apparatus, the normal varia- 
tions in cerebrospinal pressure are between 50 and 300 milli- 
meters of the fluid itself or between 5 and 7.5 millimeters Hg. 
By careful experimentation and elaborate apparatus, the normal 
variations have been found to lie between 120 and 180 milli- 
meters of distilled water. 

Physiologic Modifications. — Crying or coughing during the 
examination will cause a rise in pressure of 50 millimeters 
or more. Posture also influences pressure, as is shown if the 

3 Abstract of article by Francis Peyton Rous, M.D., in International Clinics, 
vol. ii, Seventeenth Series. 



CYTOLOGIC STUDIES. 381 

patient (who should be reclining horizontally on one side) raises 
his head. The column further shows fluctuations synchronous 
with the pulse and respiration. Finally the absolute pressure 
will be higher than normal if the patient exerts any muscular 
force during the examination. Muscular exertion should be 
eliminated as much as possible during the reading. 

Pathologic Modifications. — Marked increase in pressure is 
the rule in hydrocephalus, in brain tumor, and in meningitis of 
bacterial origin, especially when the infection is acute. Increase 
in pressure often accompanies uremic coma. A column of 600 
to 700 millimeters of fluid is not uncommon in any of these con- 
ditions. Low pressure is observed in infants in conditions giving 
low blood-pressure (see section on "Clinical Hematology'^), and 
when an error in technic has occurred. 

Collection of the Specimen. — AVhen the pressure has been 
determined, disconnect the manometer and collect a few cubic 
centimeters in a sterile capillary tube as the fluid emerges from 
the needle. This specimen should be preserved under aseptic 
precautions for bacteriologic examination; from 4 to 6 cubic 
centimeters more should be allowed to run directly into a clean 
centrifuge tube for cytologic examination. 

CYTOLOGIC STUDIES. 

Determination of the Cell-Content. — In the normal cerebro- 
spinal fluid there are from one to seven white cells per cubic 
millimeter. Probably they are never entirely absent. When the 
meninges are sound none but lymphocytes occur; these are often 
much degenerated. The fluid is normally clear, and even if it 
contains several hundred cells per cubic millimeter, it may re- 
main clear macroscopically. 

The cells may be counted with the Thoma-Zeiss hemo- 
cytometer, using the "red" pipette for measuring. Since there 
is usually some hemorrhage accompanying the puncture, it is 
necessary to have previously counted the number of red and 
white cells in the patient's blood to determine the ratio of these 
cells in the circulation, so that after counting the red and white 
cells in the cerebrospinal fluid it will be possible to determine 
how many white cells are adventitious (due to hemorrhage). 
To determine the cell-content of the cerebrospinal fluid it is only 



382 BODY FLUIDS, EXUDATES, TRANSUDATES, SECRETIONS. 

necessary to place a drop of the freshly drawn fluid upon the 
depression in the chamber and apply the cover-glass; to count 
the red cells one filling will be all that is necessary. For the 
white cells, if the cells are scant, five or ten complete fields 
must be counted and an average obtained. 

Example. — Suppose in the specimen we find 80,000 red 
cells and 102 white cells per cubic millimeter, while in the blood 
itself there are 4,000,000 reds and 5000 whites, or a proportion 
of 800 to 1. Calculating on this ratio of the 102 white cells 
present per cubic millimeter of the cerebrospinal fluid, 100 are 
from the contamination blood. Thus, but two white cells per 
cubic millimeter were present in the fluid before contamination 
by hemorrhage. 

Method of Baybee and Lorenz. — In the method of Bay- 
bee and Lorenz^ the cells are counted in a Thoma-Zeiss blood- 
counting chamber, in which the spinal fluid is first mixed in the 
red blood-counting pipette with a special diluting fluid (see 
Appendix, page 504). 

The te clinic is as follows : Draw the diluting fluid up into 
the pipette to division 7, remove the tip of the pipette from 
the diluting fluid, then draw the contents of the capillary por- 
tion of the tube up into the counting chamber coating the sides 
of same. The pipette is then filled by drawing up fresh puncture 
fluid and shaken five minutes, allowed to stand fifteen to twenty 
minutes, shaken again, and counted. 

Patholo^c Variations in the White Cells. — The cell-content 
of the cerebrospinal fluid is an extremely sensitive index of the 
state of the meninges. 

Acute Meningitis. — Purulent fluids may contain from 
4000 to 40,000 white cells per cubic millimeter. 

Tubercular meningitis will average between 200 and 300 
cells per cubic millimeter, although it may be as high as 20,000. 

Syphilitic meningitis^ tabes, paresis, usually give more 
than 10 and less than 100 cells per cubic millimeter. 

Caution. — It must be borne in mind that the occurrence of 
a slight increase in the number of white cells may be the result 



* Arch. Int. Med., vol. vii. 



CYTOLOGIC STUDIES. 383 

of a previous puncture. This question should be ascertained 
before arriving at final conclusions in any case. 

It must be remembered, further, that in cerebrospinal fluid 
kept at room-temperature for a short time the cells rapidly de- 
generate so that they are often unrecognizable after a lapse of 
a few hours, even if they have not entirely disappeared. 

Differential Count. — In general, should the pressure of the 
fluids be between normal limits, and the cells less than 10 per 
cubic millimeter, it is unnecessary to make a differential count. 
Under these circumstances lymphocytes alone are present. 
The differential count should be accomplished speedily after 
removal of the fluid to prevent alteration due to decomposition. 
The fluid which has been allowed to fall into the centrifuge tube 
should be revolved rapidly for from ten to thirty minutes. This 
completed, the tube should be carefully removed from its metal 
sheath to avoid dissipating the cells. To transfer the sediment 
to a slide or cover-glass the fluid must be drained away by 
slowly and steadily inverting the tube. While the tube is still 
inverted the slight portion of fluid remaining in the end of the 
tube is taken up on a capillary pipette and blown out on to the 
previously cleaned glass. This is air-dried, when it is ready for 
fixing or staining (for methods of staining for differential count 
see section on "Clinical Hematology"). 

Lymphocytes alone (and perhaps occasionally a large, flat 
endothelial cell) are normal to the fluid. In the differential 
count from the stained specimen, it is necessary to count and 
classify the polymorphonuclears, the mononuclears, and endo- 
thelial cells. Mast cells, eosinophiles, tumor or nerve cells, have 
rarely been detected, and must still be considered exceptional, if 
not doubtful. The relationship of the polymorphonuclears and 
the lymphocytes is alone of importance in our present state of 
knowledge. 

Polymorphonuclears are indicative of an acute process, 
while the mononuclears speak for chronicity. The mononuclears 
(lymphocytes of blood) appear to be particularly numerous in 
tubercular meningitis and cerebrospinal syphilis. 



384 BODY FLUIDS, EXUDATES, TRANSUDATES, SECRETIONS. 

CHEMICAL EXAMINATION. 
Determination of Protein Content. 

The fluid remainiiig after the bacterial examination, and 
that supernatant after centrifugation, may be devoted to the 
proteid determination. An albumin and a globulin have been 
found in the cerebrospinal fluid; for clinical purposes their 
combined content usually is determined. The acetic acid boiling- 
test may be applied here, provided the examiner is familiar with 
the normal amount of proteid as represented by the white cloud 
formed in the upper half of the tube. For a rough quantitative 
determination a very narrow test-tube may be marked ofl in 
lengths to correspond to those of the Esbach tube. The Esbach 
reagent is used as in the similar test for albumin in urine. This 
test will serve to show the proteid variations from time to time 
in a given case. 

Eoss-JoNES Test. — The technic of the Eoss-Jones test is 
a refinement of the above and is of greater value, because it 
demonstrates an increase in the globulin content, which fails to 
react to this test when in normal amount. The test: 2 cubic 
centimeters of a saturated solution of pure ammonium sulphate 
(saturated by the aid of heat) are placed in a test-tube and over 
this is laid very carefully 1 cubic centimeter of cerebrospinal 
fluid. A positive reaction is shown by a white ring which forms 
at the zone of contact within three minutes. This ring is best 
seen by indirect illumination before a dark background. 

NoGUCHi^s Butyric Acid Test. — One part (0.1 or 0.2 cubic 
centimeter) of spinal fluid is mixed with 5 parts (0.5 cubic 
centimeter) of a 10 per cent, butyric acid solution (only chem- 
ically pure may be used) in physiologic salt solution. This 
mixture is heated to boiling and immediately 1 part (0.1 cubic 
centimeter) of normal (4 per cent.) sodium hydrate solution 
is added and the mixture again boiled for a few seconds. A 
positive test is shown by a definite flocculent precipitate which 
occurs immediately or within two hours. A faint cloudiness 
may be seen in normal fluids. The fluid tested must not contain 
blood. The test is of decided clinical value. It appears regu- 
larly in the cerebrospinal fluid of patients with syphilitic and 
parasyphilitic affections and also in all cases of inflammation of 



DETERMINATION OF PROTEIN CONTENT. 385 

the meninges caused by such organisms as the meningococcus, 
pneumococcus, influenza bacillus, tubercle bacillus, etc. These 
latter cases are, however, easily differentiated from the syphilitic 
affections. Normal fluid gives a turbidity, but the granular 
precipitate does not occur at all or only after many hours. 

Albumin Estimation of Cerebrospinal Fluid. — This 
method, used by J. Gr. Greenfield,^ is a modification of Noguchi's 
butyric acid reaction, in which he uses 2 cubic centimeters of 
fluid and the reagents in the same increased proportion. The 
result of the test is then poured into a graduated centrifuge 
tube, which gives readings to 0.1 cubic centimeter with fair 
accuracy; each 0.1 cubic centimeter by this method is equivalent 
to 0.025 per cent. Small quantities of fluid can thus be ex- 
amined, and fairly reliable readings obtained from them. Green- 
field found that a 0.4 cubic centimeter precipitate corresponds 
fairly closely to 1 part per 1000. Normal fluids give readings 
of 0.05 to 0.02 cubic centimeter (corresponding to 0.1 to 0.5 part 
per 1000). Syphilitic meningitis and parasyphilitic disease give 
readings up to 0.6 cubic centimeter, or 1.5 per 1000, but in no 
uncomplicated case did Greenfield get readings above this level. 

NoNNE^s Test. — This test like the preceding tests is for the 
detection of an increase in protein content in the spinal fluid. 

There are usually described two steps or phases. 

Phase I. — Add an equal column of hot saturated solution 
of ammonium sulphate to 2 or 3 cubic centimeters of cerebro- 
spinal fluid (this must be clear). A positive reaction will be 
shown by the appearance within three minutes of an opales- 
cence or definite turbidity. The fluid reacts to this test in all 
infections of the cerebrospinal system, being of special signifi- 
cance in general paresis, obscure cerebrospinal syphilis and 
tabes. The test is chiefly valuable in its negative phase. 

Phase 11. — ^In the absence of turbidity in Phase I add a 
drop or two of acetic acid to the mixture, a distinct turbidity 
should appear at once. This may occur with normal fluids so 
little value is attached to it. 

Significance. — Although Noguchi's and the allied reactions 
are positive in many non-syphilitic conditions, yet they have a 
certain clinical value. They are not specific and, when present, 

5 Lancet, Sept. 7 and 11, No. 4045. 



386 BODY FLUIDS, EXUDATES, TEANSUTDATES, SECRETIONS. 

do not necessarily indicate a syphilitic infection. On the other 
hand;, they can be employed to establish or confirm a deduction 
based upon the clinical histor}' and the results of the Wassermann 
reaction and cyto diagnosis, thus becoming of great indirect 
diagnostic value. Thus, a syphilitic infection is practically ex- 
cluded by a negative reaction. In this respect the test has an 
advantage over the Wassermann reaction, in which a negative 
result cannot always be relied upon to indicate an absence of 
syphilitic infection. 

Lak-ge's Colloidal Gold Test. — This is also a test to de- 
termine the presence of protein material in the spinal fluid and 
is far more differential than Xog-uchfs butyric or Xonne's tests. 

Tlie Test. — (The spinal fluid must be free from blood). 
Prepare a rack with 12 narrow clean test-tubes. In the first, 
place 1.8 cubic centimeters of an 0.4 per cent, freshly made 
sodium chlorid solution. In each of the remaining 11 tubes 
place 1 cubic centimeter of the chlorid solution. To the first 
tube add 0.2 cubic centimeter of spinal fluid. The dilution in 
this tube is therefore 1 to 10. ]\Iix this thoroughly and place 
1 cubic centimeter of this dilution in the tube ^NTo. 2. This will 
give a 1 to 20 dilution. Proceed in the same way with the 
remaining tubes, which will get the dilution up to 1 to 20,440. 
After these dilutions are made add 5 cubic centimeters of the 
colloidal solution (see Appendix, page 499), to each tube and 
allow the rack to stand at room temperature for twenty-four 
hours. The test at the end of twenty-four hours reads as 
follows : — 

Tube 1 originally had a magenta shade. As the precipita- 
tion of gold occurs the color in the tubes becomes bluer and 
finally disappearing leaves them clear and colorless. The effect 
on the various dilutions are expressed by the — and -}" sig-ns. 

This — indicates no precipitation == deep red 

-\- red with beginning blue tinge. 

-| — ^ red-blue and blue-red shades. 

-| — [—\- violet and blue colors. 

-| — \--\ — |- light blue shades. 

-\ — I — I — \ — \- clear, complete precipitation. 



ORAL SECRETIONS. 387 

In syphilitic and parasyphilitic conditions the tendency is 
to precipitate toward the low (1 to 10) dilution. It rarely 
passes dilutions of 1 to 160. In non-syphilitic cases the 
tendency is toward high (1 to 20,000) dilutions, the maximum 
usually being observed at some point above 1 to 80. 

Examination for Tubercle Bacilli. — Hemenway^ reports a 
total of 135 positive results in examining 137 cases by the 
following technic: The fluid is collected in several test-tubes, 
allowing about 20 cubic centimeters per tube. The last fluid 
withdrawn usually shows the bacilli most readily. The tubes 
must not be agitated, but placed in an incubator overnight; 
sometimes three hours will sufiice. A cobweb-like film forms, 
which is then removed by a platinum loop, spread flat upon the 
slide, and fixed and stained as usual for the tubercle bacillus. 
In about 4 per cent, of fluids the coagulum does not form. In 
such cases scraping from the sides of the test-tube mounted 
on slides often show the bacillus. The length of time necessary 
for search averages one hour, but thirty to forty minutes are 
generally rewarded by positive findings. For other bacteriologic 
and staining methods, see section on Bacteriology, Chapter XII, 
pages 407 to 437. 

ORAL SECRETIONS. 

General Considerations. — The oral fluid is a mixture con- 
taining the secretions of the various buccal glands, the submax- 
illary, sublingual, parotid, and mucous glands. This secretion 
is called saliva. It is a colorless, odorless, and tasteless fluid, 
usually somewhat stringy and frothy and which separates on 
standing into two layers, the upper one of which is clear and the 
lower one cloudy. 

Chemical Characteristics. — The normal daily amount of 
saliva secreted is estimated to be about 1500 cubic centimeters; 
this quantity, however, varies greatly. The specific gravity 
ranges between 1002 and 1009. The total solid content is from 
2 to 12 grams. Its reaction is mildly alkaline. While the re- 
action of the saliva is normally always alkaline, we occasionally 
find an acid reaction, especially in children and in the early 

6 Amer. Jour. Dis. of Children, vol. 1, 1911. 



388 BODY FLUIDS, EXUDATES, TRANSUDATES, SECRETIONa 

morning hours, due to the production of lactic acid by the bac- 
teria which are always present in the mouth. Likewise, we find 
an acid reaction, especially in conditions associated with acidosis. 

The presence of potassium sulphocyanate (KCNS) is more 
or less characteristic of normal saliva and may be detected as 
follows : Collect a few cubic centimeters of saliva before a meal 
and allow this to filter. Add a few drops of hydrochloric acid 
and then a drop or two of ferric chlorid solution, when a distinct 
red color will develop, the depth of which will depend upon the 
amount of sulphocyanate present. 

Ptyalin. — -The most important constituent of saliva is the 
ptyalin. This is a definite hydrolytic ferment, converting starch 
into maltose through the intermediate stages of erythrodextrin 
and achroodextrin. This action may be readily seen by treating 
a little starch paste with a few cubic centimeters of filtered saliva 
and placing the vessel in the incubator for ten to fifteen minutes. 
If at the end of this time iodin solution is added, a distinct red 
color, instead of the characteristic blue, will appear, or else an 
entire absence of color change will be noted. 

Saliva Test for Pancreatic Function. — A further re- 
finement of this reaction has been suggested by Fedeli and 
Eomaneli''' after the demonstration of Simon that saliva, 
inhibited in its special activity by the gastric juice, recovered its 
specific properties when transferred to an alkaline medium and 
a little unmodified saliva or pancreatic juice added. The test 
is not only qualitative, but also gives a quantitative estimate of 
the proportion of pancreatic juice present. The test is made 
with 1 cubic centimeter of the individual's saliva mixed with 5 
cubic centimeters of gastric juice or an equal amount of a 2.5 
per cent, solution of hydrochloric acid. After thorough mixing 
and an interval of rest of about half an hour, add 4 cubic centi- 
meters of a 1 per cent, solution of sodium carbonate to render 
the mixture slightly alkaline, and then add .20 cubic centimeters 
of a 10 per cent, starch paste. The whole is then kept in the 
incubator for two hours, at a temperature of 37° C, shaking 
up the mixture at intervals. It is then examined for the pro- 
portion of sugar that has been produced, and then 10 cubic 



7 Riforma Medica, September 13, xxv. No. 37. 



ORAL SECRETIONS. 389 

centimeters of an emulsion of fresh feces (1 part stool to 3 parts 
distilled water) are added to the mixture, which is then replaced 
in the incubator, shaking it up occasionally. After twelve hours 
the qualitative and quantitative determination of the sugar that 
has been produced testifies to the intensity of the pancreatic 
functioning. The variation in the findings in regard to starch 
digestion in the first and second examinations gives an oversight 
of the conditions in respect to the saliva and pancreas function- 
ing when compared with the findings in health. 

Microscopic E'xamin.ation. — On allowing saliva to stand it 
separates into two distinct layers; the upper one is clear and 
contains the liquid portion, while the lower is cloudy and con- 
tains the morphologic elements. A microscopic examination of 
a specimen of saliva that has been allowed to sediment will show 
many epithelial cells in the form of large, irregular, squamous 
plates, derived from the mucous membrane of the mouth and 
tongue. The characteristic cells of the saliva are the salivary 
corpuscles, which resemble the leukocytes, but are larger and 
more granular. Occasionally red blood-cells are seen, but these 
have no special significance, as they are derived from ulcerative 
or irritative conditions somewhere in the mouth or the naso- 
pharynx. Many micro-organisms and mold, yeast, and thread 
types are always present. Few of these have any direct signifi- 
cance, although the Spirochceta huocalis should be borne in mind, 
especially when an examination is being made of a mucous patch 
for the Spirochceta pallida. The former is differentiated from 
the latter by the fact that its ends lie upon a line drawn longi- 
tudinally through the center of its spirals, while such a line 
drawn through the pallida lies above and below its ends. (See 
section on parasites.) On the other hand, many pathogenic 
bacteria have been found in the mouth of the healthy subject, 
such as those of pneumonia, diphtheria, and Vincent's angina, 
as well as various forms of strep toccoci and the organism of 
thrush. 

Diphtheria. — One of the most important examinations of 
the oral cavity consists of the detection of the diphtheria 
bacillus (Klebs-Loffler bacillus), as an early diagnosis of this 
disease frequently enables the physician to institute early anti- 
toxin treatment. Such an examination should never be omitted 



390 BODY FLUIDS, EXUDATES, TRANSUDATES, SECRETIONS. 

in any case of suspected sore throat, especially when any mem- 
branous patches are present. (See also page 428.) 

Vincent's Angina (Ulceromembeanous Angina). — In 
this condition smears taken from the throat, as well as the free 
saliva, will be found to contain many organisms of two charac- 
teristic types, the spirilla and the long, fusiform bacilli. Usually 
both of these t3^pes are found together, but occasionally the 
spirilla are absent. The spirilla usually measure from 36 to 40 
microns in length and % micron in breadth, while the bacilli 




Pig. 73.— Organisms of Vincent's Angina Showing Spirillum 
AND Fusiform Bacillus. 

are 6 to 12 microns in length and are somewhat thicker in the 
center than at the end. These organisms may be readily stained 
with Loffler's methylene-blue, gentian-violet, or dilute carbol- 
fuchsin; they deodorize with Gram's method. (See Fig. 73.) 

Thrush. — ^This is a condition most commonly seen in chil- 
dren, but may occur in adults, especially in those with tuber- 
culous tendencies. The saliva in this condition is usually acid 
and somewhat increased in amount. Microscopic examinations 
of the membrane show many epithelial cells, leukocytes, and 
much granular detritus, with a network of branching, thread-like 
formations, showing distinct segments. This organism is known 
as the Oidium albicans. It stains well with the ordinary aqueous 
methylene-blue solution. 



TRANSUDATES AND EXUDATES. 391 

NASAL SECRETIONS. 

Normally the nasal secretion is comparatively scanty. It is 
clear, tenacious, odorless, salty in taste, and alkaline in reaction. 
It is largely composed of mucns, shows squamous and ciliated 
epithelium in abundance with occasionally leukocytes, large 
numbers of bacteria, and Charcot-Leyden and triple phosphate 
crystals. This secretion does not present any points for study 
and seems to be of pathologic significance only in infectious 
conditions. 

THE CONJUNCTIVAL SECRETIONS. 

Under normal conditions the secretion of the conjunctiva 
and of the lachrymal gland are of little concern. In inflam- 
matory conditions of the conjunctiva we find certain organisms 
which require identification in order that proper treatment may 
be instituted and the proper prognosis given. The pseudodiph- 
theria bacillus is practically always found in made smears from 
the conjunctival secretion, yet it is rarely, if ever, pathogenic in 
this situation. 

Trachoma. — A great deal of attention has lately been 
given to some bodies which are found in trachoma, and which 
are probably the long-sought cause of this infectious disease of 
the conjunctiva. These organisms are known as the Prowazek- 
Graeff bodies. They are best stained by the Giemsa stain (see 
Appendix, page 481), smears being made in the usual manner. 

GoNOCOCCUS. — This organism may easily be found and 
identified in ophthalmia of this origin according to the methods 
outlined on page 429. 

Koch- Weeks Bacillus. — ^This organism is the cause of 
true pink eye or specific conjunctivitis. 

TRANSUDATES AND EXUDATES. 

Greneral Considerations. — The serous membranes are nor- 
mally kept moistened by liquids whose quantity is insufficient, 
except in a few instances, for a complete chemical analysis to be 
made of them. Under pathologic conditions an abundant tran- 
sudation may occur and produce accumulations of fluid in the 
serous cavities, in the subcutaneous tissues, or under the epider- 
mis. Such accumulations of fluid are known as transudates, and 



392 BODY FLUIDS, EXUDATES, TRANSUDATES, SECRETIONS. 

their composition is similar to the lymph, being, as a rule, poor 
in cellular elements and yielding little or no fibrin. These tran- 
sudates should be sharply differentiated from the accumulations 
of fluid in the same localities, which are the direct result of 
inflammatory processes in the membranes lining the serous 
cavities and which are known as exudates. Exudates are gen- 
erally rich in cellular elements and contain relatively more 
albuminous substances. 

The formation of true transudates is largely a question 
of filtration and is controlled by the rate of blood-flow, blood- 
pressure, irritation of the capillary endothelium, and the variable 
permeability of the endothelial cells. We should expect, there- 
fore, that the passage of dissolved substances from the blood 
would be regulated by the same laws that control the secretions 
of physiologic fluids, namely, the laws of passage of fluids 
through semipermeable membranes. The crystalloids would be, 
therefore, in approximately the same concentration as in the 
blood-plasma, while the colloids would be present in small 
quantities, the actual content being influenced by the special 
membrane through which the fluid passes. The condition of the 
blood would, hence, affect the chemical composition of such 
transudates, hydremic plasma yielding a fluid poorer in solids, 
while anhydremic blood would cause a transudate of higher 
specific gravity. 

From a clinical standpoint a differentiation between tran- 
sudates and exudates is frequently impossible, so that it is advis- 
able to resort to aspiration of the fluid and to the chemical 
and microscopic examination of the material withdrawn. The 
important divisions of such examinations are: (1) the chem- 
ical and physical properties of the fluid; (2) the bacteriologic 
content, and (3) the morphologic characteristics of the cellular 
elements. 

Obtaining the Specimen. — Whenever the fluid is to be 
withdrawn, either for diagnostic or therapeutic purposes, it is 
necessary to resort to puncture of the cavity containing the fluid. 
In all cases the site of puncture must be as carefully prepared as 
in any surgical procedure. Puncture is preferably made with 
a salvarsan needle with a rather large lumen, which should be 
carefully sterilized before use. 



PHYSICAL AND CHEMICAL PROPERTIES. 393 

PHYSICAL AND CHEMICAL PROPERTIES. 

Transudates are, as a rule, serons in character, usually 
transparent, colorless, or light yellow in color, but at times milky, 
reddish, or greenish, the latter practically always being observed 
after the fluid has stood exposed to the air. Such solutions are, 
as a rule, dichroic (yellow by transmitted light and green by 
reflected light). They are alkaline in reaction, have a specific 
gravity, which varies from 1006 to 1018, while serous exudates 
from the same cavities show a much higher specific gravity. 
The variations in specific gravity depend largely upon the 
amount of albumin present in the transudate, this practically 
never being over 3 per cent, and usually much lower. The chief 
proteins present are albumin and globulin, these being related 
to one another in the transudates as 1% to 1, while in the 
exudates the globulin is relatively much increased. The tran- 
sudates from the pleura contain the largest percentage of albu- 
min, while edematous fluids rarely show over 1 per cent. Tran- 
sudates do not coagnilate spontaneously, while exudates frequently 
do. Glucose is present both in transudates and exudates in 
amounts varying between 0.04 and 0.1 per cent. The mineral 
constituents of transudates are somewhat higher than in the 
exudates, the former averaging 0.96, the latter 0.89, per cent. 
Pathologically, fat, blood, uric acid, and biliary pigment may 
find their way into both types of fluid. In diabetes an excess 
of sugar and the presence of acetone bodies may be detected. 
(Tests for these substances may be found by consulting the 
index.) 

The exudates are usually straw- or lemon- yellow in color, 
depending on the degree of inflammation, but may range from 
a deep red (hemorrhagic) to a milky (purulent) shade. Biliary 
pigments may cause a bright green color, while various medica- 
ments, such as methjdene-blue, may produce a greenish-blue 
coloration. The specific gravity is almost always over 1018, the 
reaction alkaline, the albumin content usually above 3 per cent., 
reaching as high as 7 per cent., while the globulin is relatively 
much increased in comparison with albumin. The globulin 
increase is largely due to para-euglobulin. Nucleoprotein is 
especially abundant in purulent exudates in which the autolytic 



394 ^OBY FLUIDS, EXUDATES, TRANSUDATES, SECRETIONS. 

processes are more or less marked. The total nitrogen of the 
various fluids varies from 0.22 to 1.38 per cent. 

The following simple chemical tests have been suggested 
for use in differentiating an exudate from a transudate. While 
they have not yet been proven absolutely reliable, they are of 
value when taken in consideration with other clinical methods 
of the several tests recently advocated. To determine this often 
difficult point, the two following tests may be emplo3'ed: — 

Acetic Acid Test. — Pieper^ says that if 100 cubic centi- 
meters of water to which 2 drops of glacial acetic acid have been 
added are placed in a graduated cylinder and the fluid obtained 
from a puncture is instilled drop by drop, the drops will melt 
away in several milky streaks resembling cigarette smoke, dis- 
tinctly visible against a dark background, if they are from an 
exudate, while if they are from a transudate they will produce 
no cloudiness, or in rare cases a very faint, gray opacity that 
can be recognized against a dark background, but will never 
show the milky streaks described above. If the fluid obtained by 
puncture is turbid it should be filtered before the test is made. 

Modified Heller's Test. — S. G-angi^ describes a test 
which he has found simple and reliable for this purpose. It is 
based on Heller's test for albumin in the urine : In a test-tube 
containing 2 or 3 cubic centimeters of hydrochloric acid, 3 or 
4 cubic centimeters of the fluid to be examined are allowed to 
flow down the side. In the presence of an exudate a white cloud 
appears and forms a ring at the zone of contact between the two 
liquids. From the upper surface of this ring white clouds gather 
and rise, similar to the puff of smoke that rises when a lighted 
cigarette is struck lightly. These clouds gather to form a zone, 
at first at the surface, but later gradually sinking somewhat be- 
low the surface of the fluid in the test-tube, with a zone of limpid 
fluid above and below them down to the white ring. The ring 
gradually increases in width and density and finally clings to 
the walls of the test-tube and bubbles of gas are observed. With 
a transudate, on the other hand, the ring at the zone of contact 
is small and narrow and the fluid above persists limpid through- 
out or there may be no ring ; after a period of turbidity the fluid 



8 Munch, med. Woclien., January 4, 1910. 

9 Riforma Medica, Naples, September, xxv. No. 38. 



VARIETIES OF EXUDATE. 395 

mixes with the acid below and forms a homogeneous, turbid, 
brownish fluid. 

VARIETIES OF EXUDATE. 

Serous Exudates. — These are clear, of a light straw color, 
and show a specific gravity above 1018. There is a large 
amount of fibrin, as shown by the dense network microscopically, 
which contains a few red cells, probably derived from the bleed- 
ing at the point of puncture; a few leukocytes, which may vary 
in type according to the kind of bacteria causing the infection, 
and large endothelial cells from the serous membrane lining the 
cavity. If the blood-cells be present in sufficient numbers to 
give a distinct red color to the fluid, it is termed a hemorrhagic 
exudate, while if a few pus-cells are found it may be called a 
seropurulent type. 

The type of leukocytes present is usually of the polymor- 
phonuclear variety, although other forms may be present in small 
numbers. 

Chylous Exudates. — Such exudates show all the properties 
of chyle. The fluid is white and milky, contains between 1.5 
and 2.5 per cent, of protein, and a considerable amount of fat, 
which may be demonstrated by staining with osmic acid or 
Sudan III or by alkalinizing with sodium hydrate and shaking 
out with ether. 

Hemorrhagic Exudates. — This type is, in reality, a sero- 
fibrinous form containing large numbers of blood-cells. It is 
observed in patients with hemorrhagic diathesis, in connection 
with active tuberculosis, with neoplasms of the serous cavities, 
and following injuries to the chest or abdomen. In this form 
of exudate, while usually due to the tubercle bacillus, the 
organism is only occasionally found. The type of leukocytes 
(mononuclear) would be very strong presumptive evidence in 
favor of tuberculosis, even though no bacilli were present. If 
the exudate be due to a malignant growth, shreds of the tumor 
tissue, if found, may make a probable diagnosis. 

Purulent Exudates. — ^These are composed either of true pus 
or of seropus. They are yellowish in color, thick, and occasion- 
ally tenacious, separating on standing or centrifuging into a 
cellular deposit and a pus-serum. The cells forming the pus 



396 BODY FLUIDS, EXUDATES, TRANSUDATES, SECEETIONS. 

are not infrequently in a condition of advanced fatty degener- 
ation and may contain numerous bacteria. The addition of 
dilute acetic acid will usually clear up the cells and render the 
nuclei recognizable. 

Putrid Exudates. — These may be observed in various cavi- 
ties of the body or in the substance of various organs, especially 
the liver and lungs. The material obtained by puncture is 
usually brownish or greenish in color, has a very offensive odor, 
and is usually alkaline, but may be acid. Microscopically, de- 
generated cells, numerous bacteria, cholesterin, fatty acid, and 
hematoidin crystals are observed. Bilirubin crystals and various 
amino-acids may be found in rupture of a hepatic abscess. 

MICROSCOPIC EXAMINATION. 

General Considerations. — The cytology of transudates and 
exudates has reference to the study of the various types of cells 
found in such fluids. As a rule, these investigations are most 
frequently carried out on the non-purulent fluid, as in the ex- 
aminations of the purulent fluids we are generally concerned with 
the bacteria present. 

Preparation of the Specimen. — The fluid when obtained 
should be promptly centrifuged before the formation of any clot. 
If it is impossible to examine it promptly, the clot should be 
broken up by shaking the fluid in a small flask which contains 
a few glass beads. Small Erlenmeyer flasks should be sterilized 
at 150° C. and kept ready for use. It has been found that the 
proportion of leukocytes obtained by prompt centrifugalization, 
and also after defibrinization, is practically the same. 

It is necessary to centrifuge the fluid because of the small 
number of cells which are usually present, the concentration of 
the exudates rendering the examination much more rapid and 
satisfactory. This is especially true of serofibrinous pleuritic 
and peritoneal exudates, as but few elements are usually present 
in such exudates. If possible from 15 to 20 cubic centimeters 
should be withdrawn, though fair results can be obtained from 
1 to 2 cubic centimeters. 

The fluid should always be examined within twenty-four 
hours, as otherwise changes take place in the morphology of the 



CYTOLOGY OF FLUID EXUDATES. 397 

leukocytes which render recognition exceedingly difficult. All 
fluids for such examinations should be kept on ice to inhibit 
bacterial growth. 

Centrifugalization should be prolonged (ten or more min- 
utes), but not too rapid, for fear of destroying beyond recognition 
already partially degenerated elements. 

Cytology of Normal Fluids. — The number of cellular ele- 
ments in fluids from the various serous cavities of the body may 
vary from a very few to a large number. The cells observed are 
the red and white corpuscles of the blood, the latter of which 
are usually relatively more numerous than in the circulating 
blood and are usually largely of the polynuclear type, although 
mononuclear forms are frequently present. Neutrophiles and 
eosinophiles are present under normal conditions, the latter 
being relatively more abundant than in the blood. If a large 
number of red cells are found, an injury of the small vessels 
during the puncture usually explains their presence. Besides 
these types of cells, which are similar to those of the blood, a few 
endothelial cells from the lining of the cavity will usually be 
found. 

In examining normal as well as pathologic fluids for their 
cellular contents 100 cells should be counted if possible and the 
percentage of each type thus determined. This constitutes the 
cytologic formula of the exudate. Such examinations may be 
carried out according to the technic outlined on page 58, using 
the Zappert apparatus. 

Cytolo^ of Pathologic Fluids. — The predominant cell in 
most infective processes is the polymorphonuclear neutrophile; 
the two exceptions to this are found in tuberculous and typhoid 
infections, when the lymphocyte is relatively much increased. 
This is often a valuable differential point in the study of such 
exudates. 

THE PERITONEAL FLUID. 

Characteristics of Normal Fluid. — In conditions of health 
there is just sufficient fluid to thoroughly lubricate the interior 
of the abdominal cavity. This fluid is clear, of a pale, straw 
color, having a specific gravity of 1005 to 1015; is slightly 
albuminous and, under the microscope, a drop of freshly drawn 



398 BODY FLUIDS, EXUDATES, TRANSUDATES, SECRETIONS. 

fluid placed between a clean slide and cover-glass reveals very 
few, if any, formed elements. 

Pathologic Exudate. — Inflammatory ascitic fluid is 
straw or lemon-yellow in color, and usually somewhat cloudy, 
depending upon the number of cells contained in it. Its specific 
gravity varies between 1012 and 1026, or even higher. The fluid 
frequently coagulates spontaneously. It shows a variable amount 
of albumin by boiling, and sugar, bile-pigments, urea, uric acid, 
and cholesterin may be demonstrated by appropriate means (see 
section on "Urine^'). More rarely xanthin, creatinin, and allan- 
toin may be found. 

Hemorrhagic effusion occurs in cancer and tuberculosis 
of the abdominal cavity, and occasionally in cirrhosis of the 
liver. 

A CHYLOUS or MILKY FLUID is Occasionally met. This is 
readily distinguished by examination of fresh fluid under 
the microscope, when the characteristic fat globules will be 
found. 

Won-inflaminatory Transudate. — In Bright's disease the 
ascitic fluid is usually a pale clear serum of low specific gravity, 
showing a minimum of cells. 

In cirrhosis of the liver the color is usually darker and bile- 
pigment can be demonstrated. 

CYST FLUIDS. 

Differentiation. — Ovarian Cysts: The fluid obtained 
from ovarian cysts has a specific gravity usually above 1030, and 
is of a dark-browny g:^iimous appearance. Ovarian fluids are fur- 
ther said to contain a large number of compound granule cells 
which, from their frequent occurrence in this fluid, have been 
termed ^^ovarian cells.^^ Microscopically these appear as large 
oval or round cells, showing dense, irregular granulations often 
obscuring the nucleus of the cell. 

EcHiNOCOCCUS Cysts. — The fluid obtained by puncturing 
an echinococcus cyst is, in uncomplicated cases, perfectly clear, 
free from albumin, and contains a characteristically large 
amount of sodium chloride, which can easily be identified by 
allowing a drop of the fluid to crystallize out on a slide and 
examining for the square crystals of the salt. The specific gravity 



PLEURAL FLUID. 399 

varies between 1008 and 1013. Very rarely, no trace of any 
morphologic elements can be found. Occasionally, only a few 
hemosiderin or cholesterin crystals and a few fatty cells are 
present. Usually, however — and this is the most important point 
— ^we find, in snch fluid, remnants of the scolices of the echino- 
coccus. When these are absent, a careful search of the fluid will 
frequently reveal the presence of small booklets derived from the 
scolices. These booklets furnish an absolute diagnosis of the 
presence of the echinococcus embryos. 

Pancreatic Cysts. — ^The puncture fluid which is obtained 
from pancreatic cysts varies greatly in its physical properties, 
depending upon the antomical nature of the cyst and the length 
of time the exudate has remained in the cyst cavity. The exu- 
dates which have been rapidly formed, either in traumatic cysts 
or in cysts connected with malignant new growths of the pan- 
creas, are usually hemorrhagic and have a high specific gravity 
—that is, from 1020 to 1030. 

Ferments are present in the cyst contents and may be used 
to identify the fluid when the exudate has existed but a short 
time. The contents of old cysts rarely give evidence of the 
presence of either the proteolytic or fat-splitting ferments. 

If the fluid from a suspected cyst digests egg albumin disks 
or fibrin, or, as suggested by Boas, acts upon the proteids of milk, 
the presence of pancreatic secretion in the cyst contents may be 
considered probable. For the milk-test, mix the fluid with fresh 
milk, heat to 37° C. for several hours, precipitate the casein with 
acetic acid, and test the fluid by the biuret reaction. This test 
should always be controlled by a parallel one, using milk without 
cyst fluid. 

PLEURAL FLUID. 

General Considerations. — Physiologically there is not pres- 
ent enough fluid for analysis. Under pathologic conditions it 
may vary from one to four or more liters, and may be serous, 
sero-purulent, purulent, or hemorrhagic. 

Non-inflammatory Transudate. — In hydrothorax the spe- 
ciflc gravity, as a rule, is below 1015, the albumin content is 
small, and there is little tendency for the fluid to coagulate spon- 
taneously. The fluid is clear or pale, straw-colored, unless 



400 BODY FLUIDS, EXUDATES, TRANSUDATES, SECRETIONS. 

tinged with hemorrhage occurring during the puncture. The 
formed elements are very scarce and hard to demonstrate. 

Inflammatory Exudates. — Seeo-fibrinous Pleurisy : The 
serous exudate is abundant, and flakes of fibrin are present in 
the fluid, appearing as fibrillated flocculi. The actual amount of 
fluid varies greatly. It is of a citron color, either clear or slightly 
turbid from the fibrin and formed elements present. In some 
cases the color is dark-brown, usually due to altered blood con- 
tained in it. Specific gravity below 1020. 

Microscopically/, we find a variable number of leukocytes 
and red blood-cells, and some swollen endothelial cells and bac- 
teria. 

Purulent Pleurisy. — The specific gravity is usually above 
1020. The fluid has a heavy, sweetish odor, which, if the in- 
fection be due to a penetrating wound, may be fetid. The fluid 
is essentially pus, contains a very large amount of albumin which 
may coagulate spontaneously after removal. Appropriate tests 
may show cholesterin, uric acid, bile-pigment, and sugar. 

Method of Making Permanent Stained Preparation. — In 
fluids which are clear and show but few cells on microscopic 
examination of the fresh specimen, it becomes necessary to cen- 
trifuge 15 to 20 cubic centimeters in order to concentrate 
these formed elements. After centrifugalization in the ordinary 
urine centrifuge for from two to three minutes, the supernatant 
liquid is slowly and steadily poured off, and the remaining resi- 
due taken up in a capillary pipette from which it is blown out 
upon clean cover-slip and allowed to dry spontaneously (without 
heating). It then may be stained by any of the Eomanowski 
stains (see section on ^^Clinical Hematology^^) or the eosin- 
methylene-blue sequence. 

Fluids which contain cells in macroscopic amount may be 
transferred to cover-glasses for staining without centrifugali- 
zation. 

Microscopic Examination of Stained Preparations. — In 
hydrothorax the cells are few and mainly large, flat, of endothe- 
lial origin. 

In pneumo- and strepto-coccic pleurisy there is a great pre- 
ponderance of polymorphonuclears. In tubercular inflamma- 
tions the lymphocyte is the predominating cell. 



THE PERICARDIAL FLUID. 401 

A large number of endothelial cells, occurring especially 
in sheets or plaques, usually means a mechanical effusion or 
transudate. 

Many efforts at classification and diagnosis have been made 
on a limited number of examinations with a view to arriving 
at some definite rules for cytodiagnosis of pleural effu- 
sions. Further systematic study along these lines is, however, 
necessary. 

The presence of red cells is usual in tuberculous processes, 
also in the presence of rupture of an abscess from an adjacent 
organ into the pleural cavity. Very frequently red blood-cells, 
in a pleural effusion, are due to the traumatism inflicted at the 
time of puncture. Eed cells which appear in the first few cen- 
timeters drawn are most likely of this origin. 

The possibility of diagnosing cancer by microscopic exami- 
nation of the fluid is always present, through the finding of 
characteristic cell masses in the fluid. 

THE PERICARDIAL FLUID. 

This fluid normally is of a pale, lemon-yellow color, slightly 
viscid, cloudy from cell detritus; occasionally it may be clear. 
It contains a moderate amount of albumin and certain inorganic 
salts. The specific gravity ranges between 1015 and 1030. 

THE SYNOVIAL FLUID. 

This fluid is alkaline, thick, viscid, sticky, and of a yellow- 
ish color. Physiologically the joints contain just sufficient to 
completely lubricate them. Under pathologic conditions the 
fluid, in a large joint, may amount to many cubic centimeters. 

HYDROCELE FLUID. 

The fluid is usually clear, of a yellow or greenish tinge. 
The specific gravity varies between 1014 and 1026. The fluid 
sometimes coagulates spontaneously. Some leukocytes are 
always present, and occasionally crystals of cholesterin. 



2G 



XII. 

HUMAN MILK. 



General Considerations. — It must always be remembered, 
in the examination of milk, that it is no simple matter to obtain 
a truly representative sample. The fatty matters tend to sepa- 
rate, and there may be great variations between different por- 
tions of the same sample. This difficulty is particularly pro- 
nounced when human milk is examined, because of changes in 
the milk incident to mental and nervous disturbance. 

The Sample. — The sample should consist of a thorough 
mixture of portions taken at different times, by means of the 
breast-pump. Wherever possible it is well to have the woman 
somewhat accustomed to the use of the pump before taking any 
milk for examination. In all cases the sample should be thor- 
oughly mixed before testing, by pouring rapidly from vessel to 
vessel. 

Physical Characteristics. — Woman's milk is bluish-white in 
color, of sweetish taste and characteristic odor. When freshly 
drawn it is alkaline or amphoteric, but never under healthy 
conditions is it acid. The specific gravity varies between 1026 
and 1036, the average being 1031 at 60° F. On the addition 
of acetic acid only a slight coagulum is seen, being in the form 
of small, fine flocculi, never in large masses as is the case with 
cows' milk. Besides the myriad fat-globules, may be seen large, 
flat epithelial cells from the milk ducts. 

Composition of Human Milk (after Holt). 



Fat 

•Sugar 

Proteids 1.50 

Salts 0.20 

Water 87.30 

Total 100.00 



Average. 
4.00 per cent. 
7.00 " 



J^ormal variations. 
3.00 to 5.00 per cent. 
6.00 to 7.00 
1.00 to 2.25 
0.18 to 0.25 

89.82 to 85.50 

100.00 to 100.00 



(402) 



EXAMINATION OF THE MILK. 403 

The composition and^ food value of human milk are im- 
paired when the mother is unhealthy. It is affected injuriously 
when there exists undue emotional excitement. It will contain 
an appreciable amount of certain medicines ingested by the 
mother, which may affect the infant. 

Examination of the Milk. — The exact determination of the 
composition of human milk is only to be determined by a com- 
plete chemical analysis. There are, however, many variations 
from the normal which the physician may readily ascertain for 
himself by simple methods of examination. 

The Quantity. — This may be determined roughly by using 
the breast-pump, although this is not reliable for many reasons. 
With sufficiently sensitive scales the physician may, by weigh- 
ing the infant before and immediately after nursing, determine 
whether it is getting only one or two, or four or five ounces. The 
average daily quantity secreted by women is one liter or two 
pints. 

The Eeaction. — Test by litmus paper. The reaction 
should be alJialine or amphoteric, but never acid when freshly 
drawn. 

A ^ spontaneous change occurs after milk has stood for some 
time in a warm place; it coagulates or sours, and becomes acid. 
This is due to the change of milk-sugar (lactose) into lactic 
acid. This occurs through the agency of the bacterium lactis. 
The casein normally is held in solution by the alkaline phos- 
phates, the acid changes the reaction, and hence causes the 
casein to be precipitated. 

Specific GtRAVitt. — This may be talcen by a small hydrom- 
eter which is graduated from 1010 to 1040 (1028 to 1034 aver- 
age variation). The specific gravity is lowered by fat, but 
increased by the other solids. 

Microscopically, milk is found to be composed of minute 
brilliant, oil globules encased in a thin envelope of casein. 
Immediately after delivery the ,milk is relatively poor in casein, 
but rich in fatty matter, which exists in considerable amount in 
the form of colostrum masses. The microscope also reveals the 
presence of colostrum corpuscles, blood, pus, epithelium, and 
granular masses. Colostrum corpuscles are abnormal after the 
twelfth day; blood and pus are always abnormal. 



404 HUMAN MILK. 

Determination of the Fat. — The simplest method is by 
means of the cream gauge. This consists of a graduated test- 
tube with a foot, and a ground-glass neck and stopper. The 
tube is filled to the zero mark with freshly-drawn milk and 
allowed to stand at room- temperature for twenty-four hours, 
when the percentage of cream is read off directly from the grad- 
uated scale. The relation of cream to fat is approximately live 
to three. Thus 5 per cent, cream equals 3 per cent, fat, etc. 

Centrifuge Method. — The use of a specially graduated 
centrifuge tube is more accurate. These tubes may be used in 
the ordinary centrifuge for urine. Only 6 cubic centimeters of 
milk are required for this test. If carefully conducted the test 
is nearly as accurate as the chemical analysis. It gives results 
accurate to within one-fifth of 1 per cent. 

In the usual apparatus two pipettes are supplied with the 
centrifuge tubes, one of 5 cubic centimeters capacity, marked 
milk, the other holding 1 cubic centimeter up to a mark, for 
introducing the alcoholic solution. 

To determine the fat by this method, 5 cubic centimeters 
of the sample is introduced into the tube by means of the 
pipette marked '^milk," 1 cubic centimeter of the alcoholic solu- 
tion (solution-A) (see Appendix, page 494), is added, and the 
tube well shaken. Then, by means of any large pipette, solu- 
tion-B (see Appendix, page 494) is added little by little 
until the tube is fiUed to the zero mark. It is then placed in 
the centrifuge and rotated very rapidly for four or five minutes. 
This will bring the fat to the top in a clear yellowish layer 
which can be read off in, direct percentage by the scale on the 
neck of the tube. 

A few drops of water may be added, if necessary, toi correct 
the level of the top of the fluid. If the milk should be richer 
than 5 per cent, it will be necessary to dilute the sample with an 
equal quantity of water^ proceed with the test as above, and 
finally multiply the result by two. 

This solution may be kept for a short while; if it turns 
dark it is worthless. Solution-B consists of sulphuric acid^ 
specific gravity 1832. 



DETERMINATION OF THE FAT. 



405 



Composition of Woman's Milk. 



Fat ... 
Sugar . 
Proteids 
Salts .. 
Water . 




Common healthy variations. 


Per cent. 


3.00 to 


5.00 


6.00 to 


7.00 


1.00 to 


2.25 


0.18 to 


0.25 


89.82 to 


85.50 


100.00 


100.00 



Average Normal Excretion. 

Approximately. 

At the end of the first week 10 to 16 oz. (300 to 500 Gm.) 

During the second week 13 to 18 oz. (400 to 550 Gm.) 

During the third week 14 to 24 oz. (430 to 720 Gm.) 

During the fourth week 16 to 26 oz. (500 to 800 Gm.) 

From the fifth to the thirteenth week 20 to 34 oz. (600 to 1030 Gm.) 

From the fourth to the sixth month.. 24 to 38 oz. (720 to 1150 Gm.) 

From the sixth to the ninth month.. 30 to 40 oz. (900 to 1220 Gm.) 

Determination of the Sugar. — The percentage of sugar is 
nearly constant, so it may be ignored in the nsnal clinical in- 
vestigation. 

Estimation of the Proteids. — Method of T. E. Boggs 'A 25 
grains of phosphotnngstic acid are dissolved in 125 cubic centi- 
meters of distilled water, and when solution is complete an 
equal quantity of dilute hydrochloric acid is added. The solution 
is stable, and keeps for months in a dark bottle. The method 
of procedure is as follows : The diluted milk is poured into an 
ordinary Esbach albumin ometer tube (reading from 1 to 7 grams 
per liter) up to the mark IT. The phosphotnngstic acid solu- 
tion is then added up to the mark R; the tube is corked and 
slowly inverted twelve times, shaking being avoided. The tube 
is then placed in a rack for twenty-four hours, and the per- 
centage read off at the level of the top of the precipitate. The 
optimum dilution for human milk is 1 in 10. If the proteid 
content is low a less dilution may be employed. Controlled by 
Kjeldahl nitrogen determinations the mean error was 0.3 per 
cent., the extreme 0.7 per cent. Temperatures of from 15° to 
25° C. and the presence or absence of cream make no difference 

1 Bull. Johns Hopkins Hosp., October, 1906, No, 187. 



406 HUMAN MILK. 

in the volume of the precipitate, which attains a minimum after 
standing twenty-four hours, and does not alter on further 
standing. 

A rough estimate may be made as follows: If we 
regard the sugar and salts as constant, or so nearly so as not 
to affect the specific gravity, we may form an approximate idea of 
the proteids from a knowledge of the specific gravity and the 
percentage of fat. We may thus determine whether they are 
greatly in excess or very scanty. The specific gravity then will 
vary with the proportion of proteids, directly, and inversely 
with the proportion of fat, i.e., high proteids, high specific 
gravity; low fat, low specific gravity. The application of this 
principle will be seen by reference to the table on page 409. 

TESTS FOR FORMALDEHYDE IN MILK. 

Hehner's Test. — To 15 cubic centimeters of concentrated 
sulphuric acid in a test-tube add 1 or 2 drops of ferric chloride 
test solution (U. S. P.) and mix. Then pour upon this, in 
such manner as not to mix the layers, the suspected milk. A 
violet color indicates the presence of formaldehyde. In the case 
of cream dilute the cream with an equal volume of water, and 
then apply the test as above described. The violet color is some- 
times produced at once, but oftener not for five or ten minutes, 
and sometimes not for an hour or so, depending on the amount 
of formaldehyde present. By this test 1 part in 10,000 or 15,000 
is readily detected. 

Liebermann's Phei^ol Test.— In the presence of small 
traces of formaldehyde, distil off from the milk a few cubic 
centimeters and add to this 1 drop of very dilute aqueous phenol 
solution. Then pour this mixture slowly upon concentrated 
sulphuric acid in a test-tube so as to form a layer. A bright 
crimson color appears at the zone of contact. This may occur 
with as little as 1 part in 200,000 and with ease in proportions 
of 1 to 100,000. There is a milky zone above the red color, and, 
if more concentrated, there will be a whitish or pinkish pre- 
cipitate. Sometimes the zone will appear, only in about one 
hour, and be %o i^ch below the line of contact. 



XIII. 
BACTERIOLOGIC METHODS. 

By a gradual process of evolution and development the 
field of clinical medicine has so enlarged its borders that to-day 
the laboratory worker no longer confines his investigations to 
examination of the blood, urine, and sputum, etc., but must pos- 
sess a working knowledge of bacteriology and be familiar with 
bacteriologic technic to the extent that he may be able to obtain, 
differentiate, and recognize the commoner pathogenic organ- 
isms. This section will confine itself to a brief outline and dis- 
cussion of the essentials of laboratory technic, and to a brief 
description of the more commonly encountered pathogenic micro- 
organisms. No effort has been made to make this section either 
exhaustive or complete, the author believing that a work of this 
type is more valuable if maintained within a small compass, 
even if the laboratory worker is occasionally driven to consult 
larger works, devoted entirely to bacteriology, in his search for 
some particular organism or technical detail. 

STERILIZATION. 

General Considerations. — Acquaintance with the funda- 
mental principles of sterilization and of disinfection are abso- 
lutely necessary to the successful performance of all bacterio- 
logic investigations. The term sterilization, as commonly 
employed, implies the absolute destruction of bacterial life by 
heat, while the term disinfection is commonly applied to accom- 
plishing the same end through the agency of chemical sub- 
stances capable of destroying bacterial life. 

Strictly speaking, the term sterilization implies the com- 
plete destruction of the vitality of all micro-organisms that may 
be present in or on the substance or substances to be sterilized. 
Such a result can obviously be accomplished by either thermal 
or chemical means, while disinfection need not, of necessit}^ de- 

(407) 



408 BACTERIOLOGIC METHODS. 

stroy all living organisms that are present, but only those having 
the power of infecting or of producing disease, and may or may 
not, as the case may be, cause complete destruction of bacterial 
life, as in sterilization. It is, therefore, possible to accomplish 
both disinfection and sterilization by either chemical or thermal 
means. 

In the laboratory the employment of these different terms 
depends upon and is governed by circumstances. It is, of course, 
essential that all culture media should not only be absolutely 
free from all bacteria, whether they are pathogenic or not, but 
also from their spores. In a word, they must be sterile. At the 
same time it is equally essential that the original chemical com- 
position and physical properties of the media should remain un- 
altered by the process. It is self-evident, therefore, that steriliza- 
tion of such substances by means of chemical agents is out of 
the question, for while this method would destroy all bacterial 
life, it would not only alter the chemical composition of the 
media, but by becoming inseparably mingled with the media, 
would, by its continued prsence, effectually prevent the growth 
of bacteria in the material for all time; that is to say, after 
having performed its function as sterilizer, it would by its con- 
tinued presence exercise its function as an antiseptic, and render 
the material useless as a culture medium. Exceptions to this 
general rule are found in certain volatile substances such as alco- 
hol and ether, which, after having performed their bactericidal 
powers, may be completely driven off by the application of heat. 

Sterilization by Heat. — Sterilization by means of high tem- 
peratures may be accomplished in a variety of ways : 1. By dry 
sterilization, which is accompanied by subjecting the articles to 
adequate degrees of heat in a properly constructed oven. 2. 
By subjecting them to the influence of live steam at 100° C. 3. 
By subjecting the substances to steam under pressure. When 
employing steam under pressure, the temperature to which the 
articles are subjected will depend upon the pressure developed — 
the greater the pressure the higher the temperature. 

Sterilization by Dry Heat. — This method has the following 
disadvantages which limit its applicability : 1. The temperature 
must be relatively high and the period of exposure long as com- 
pared to moist heat (steam). 2. The penetration of dry heat 



STERILIZATION. 409 

into substances to be sterilized is much less thorough than that 
of steam. 3. Many substances of vegetable and animal origin 
are rendered valueless by the temperature required for dry ster- 
ilization. 

As a preliminary to sterilization, glassware, especially when 
new, should be thoroughly cleansed and then cleared by soaking 
for one hour or more in the following solution: — 

Potassium bichromate 60 parts. 

Water 600 parts. 

Sulphuric acid 460 parts. 

Successful sterilization by dry heat cannot usually be accom- 
plished by a temperature lower than 150° C, and this tempera- 
ture must be continued for not less than an hour. In general, it 
may be said that dry sterilization is only suited for sterilization 
of such substances as glassware, dishes, flasks, test-tubes, pi- 



NOTE.— In the following table Holt explains the application of this principle; 
it must be borne in mind that the results obtained are only approximate and 
cannot take the place of the actual quantitative method, except as a rough 
guide to variations in breast milk. 





Specific 


Cream, 


Proteid 




gravity. 


24-hours. 


(calculated). 


Average 


1031 


7% 


1.5%. 
Normal (rich 


Normal variations . 


1028—1029 


8%— 12% 








milk). 


Normal variations . 


1032 


5%— 6% 


Normal (fair 
milk). 


Abnormal variations 


Low (bel. 1028) 


High (ab. 10%) 


Normal or slightly 
below. 


Abnormal variations 


Low (bel. 1028) 


Low (bel. 5%) 


Very low (very 
poor milk ) . 


Abnormal variations 


High (ab. 1032) 


High 


Very high (very 
rich milk). 


Abnormal variations 


High (ab. 1032) 


Low. 


Normal or (nearly 
so). 



410 BACTERIOLOGIC METHODS. 

pettes, etc., and for such metal instruments as are not injured 
by heat. 

Steam or moist heat sterilization possesses great penetrating 
power, and is much more rapid and thorough than the above 
method, and, further, it is far less likely to destroy the material 
so treated. This method should be employed for sterilizing all 
culture media, fabrics, cotton, wood, and organic material in 
general. 

Aside from the relative applicability of the two methods, 
their mode of action toward the organisms to be destroyed is 
very different. The penetrating power of steam renders it far 
more efficacious than dry heat. The spores of several organisms 
which are destroyed by exposure to steam for a few minutes, 
resist the destructive action of dry heat at a higher temperature 
for a longer period of time. 

The method of applying heat for sterilization depends 
chiefly upon the character of the substances to be sterilized. The 
application of dry heat is always continuous, i.e., the objects to 
be sterilized are simply exposed to the proper temperature for 
the requisite time necessary to destroy all living organisms and 
their spores which are either in or upon them. With steam, on 
the other hand, the articles to be sterilized are frequently of 
such a nature that prolonged sterilization would injure them. 
For this reason it has been found desirable to subject such ob- 
jects to the influence of steam intermittently for a number of 
short periods. 

The PEixciPLE involved in the intermittent method de- 
pends on the differing powers of resistance to heat displayed 
by different organisms in different stages of their development. 
During the life of many bacilli they enter a stage during which 
their resistance to both chemical and thermal agents is mate- 
rially increased. This increased power of resistance is possessed 
by the organisms when they are in the spore or resting stage. 
Some spores of certain organisms have been encountered which 
retain the power of germination after an exposure of more than 
an hour to the temperature of boiling water. This difference in 
the thermal heat-point of bacteria and their spores is taken 
advantage in the process of sterilization known as the fractional 
or intermittent method. 



STERILIZATION. 411 

As all culture media depend for their usefulness upon more 
or less unstable organic compounds, the effort of sterilization h 
to destroy the organisms in the shortest possible time by expo- 
sure to least possible amount of heat. This is accomplished by 
subjecting them to a temperature at a time when the bacteria 
are in the vegetative or growing stage. In order to develop any 
existing spores the media, during the intervals between steriliza- 
tion, should be kept imder such conditions of temperature and 
moisture as will favor the process of vegetation (room-tempera- 
ture). 

During the first application of heat the mature vegetative 
forms are destroyed, while certain spores which may be present 
resist this treatment and survive the temperature. Now the 
sterilization is discontinued and the media is allowed to remain 
for a time, usually twenty-four hours at room-temperature. 
During this time those spores which resisted the first heating 
have conditions favorable to germination. A second short ex- 
posure kills this crop of bacilli, when a second rest, followed by 
a third short exposure, kills the remaining organisms and the 
media will usually be found sterile. 

It should be remembered that while all spores which are 
present are not killed by the first exposure, still their power of 
germination may be so inhibited by the exposure to 100° C. 
that their germination is delayed, that they cannot possibly 
germinate during the twenty-four hours' intervals. 

Experiment has shown, that the fractional process gives the 
best results when the objects are subjected to the action of live 
steam (steam at ordinary atmospheric pressure) for fifteen 
minutes on each of three consecutive days, and that during the 
intervals the cultures should be maintained at a temperature 
between 25° and 30° C. The substances thus treated will re- 
main sterile for an indefinite time, provided they are not exposed 
to the re-entrance of micro-organisms. 

An occasional exception will be noted when, after careful 
treatment as above outlined, certain species of spore-forming 
bacteria will not have been entirely destroyed by this method. 
These are usually of the non-pathogenic group of the so-called 
soil organisms. 

Finally it must be born in mind that this method is only 



412 BACTERIOLOGIC METHODS. 

applicable to substances capable of presenting conditions favor- 
able to spore germination^ and that dry substances, such as in- 
struments, apparatus, or organic materials, in which decomposi- 
tion has set in, where the natural conditions favorable to spore 
germination are absent should not be treated by this method, 
but must be subjected to higher temperatures for longer periods 
of time. 

Intermittent Sterilization (at low temperature). — The 
process of intermittent sterilization at comparatively low tem- 
peratures is based upon the principle outlined above, but differs 
in the details of its application as follows : 1. It requires a 
greater number of exposures to accomplish complete sterilization. 
2. The temperature at which it is accomplished is not above 
68°-70° C. 

It is employed for sterilization of easily decomposible ma- 
terials and those which would be rendered useless by the appli- 
cation of steam at 100° C, but which are unaltered by the tem- 
perature employed. This method is applicable for sterilization 
of certain albuminous media where it is desirable to retain 
their fluid condition during sterilization and which would be 
coagulated by exposure to higher temperature. 

This process requires that the temperature employed should 
be between 68° -70° C, and that an exposure of one hour should 
be made each of six consecutive days. During the intervals the 
material is kept at a temperature between 25° -30° C. to favor 
germination of any spores that may be present. 

Direct Steam Method. — Sterilization by means of steam is 
also accomplished by what is known as the direct or continuous 
method. By this process both mature organisms and their spores 
are destroyed by a single exposure to steam at zero pressure — 
live or steaming steam. The sterilization is accomplished by a 
single exposure of one hour. 

Steam-Pressure Method (by Autoclave). — By employing 
steam under pressure we are able, by increasing the temperature, 
to materially shorten the time necessary to accomplish complete 
sterilization. By employing a pressure of approximately one 
atmosphere (15 pounds) a temperature of about 122° C. is 
obtained. This is sufficient to accomplish complete sterilization 
by one exposure of fifteen minutes. 



STERILIZATION. 



413 



When this method is employed it will occasionally be found 
that the coagulating power of gelatin is reduced, and that it 
becomes slightly cloudy, while in agar-agar a fine, flaky precipi- 
tate is noticed. For accurate time and temperature exposures 
this is a very uncertain method. Obviously the material is sub- 
ject to active temperatures during the heating up as well as 
during the cooling process, besides the actual time during which 
the maximum pressure is maintained. Also, if great care is not 




Pig. 74.— Arnold Steam Sterilizer. (A. H. T. Co.) 



observed to prevent premature opening of the autoclave, a 
sudden, rapid ebullition of the fluid,' occurs which, in the case 
of test-tubes, the media may completely boil away. 

While this method of sterilization is not well suited for 
delicate , experiments where a definite time exposure to definite 
temperature is of importance, still, for general laboratory pur- 
poses, it has much to recommend it in the way of a time saver, 
and in the certainty with which sterilization is accomplished. 
The apparatus designed to sterilize under pressure is termed an 
autoclave. 



414 BACTERIOLOGIG METHODS. 

Practical Application of the Method. — 1. The Arnold 

SteiAm Steeilizeor (Fig. 74) : This is probably the best steril- 
izer for general laboratory purposes, since it is simple and eco- 
nomic in its operation. The difference between this apparatus 
and the original sterilizer devised by Koch is that it provides 
for the condensation of the steam after its escape from the 




Fig. 75.— Autoclave. (A. H. T. Co.) 

sterilizing chamber, and returns the water of condensation auto- 
matically to the reservoir. 

2. The Autoclave. — The advantage of this method is so 
well recognized that its use has practically superseded the inter- 
mittent method with live steam, except when the temperature 
developed by the autoclave is sufficient to destroy the materials 
subjected to it. By this plan sterilization is accomplished in 
fifteen minutes bj^ exposure to steam under? the pressure of one 
atmosphere. 



THE AUTOCLAVE. 415 

The autoclave (see Fig. 75) embodies the same principle as 
the steam sterilizer. It provides for the generation of steam 
within a chamber capable of being hermetically sealed after the 
introduction of the substances to be sterilized. The chamber is 
fitted with a safety-valve arranged for regulating the degree of 
pressure. A thermometer passes through the wall and enters 
the chamber, thus allowing the temperature to be followed and 
the pressure properly regulated. 

HoT-AiR Sterilizer. — The hot-air sterilizers employed for 
this work are simply double-walled boxes of Swedish iron, having 
a double door and a copper bottom. They are fitted with proper 
openings in the walls to permit circulation of the heated air. 
The heat is obtained from the fiame of a Bunsen burner applied 
directly to its bottom. These bottoms are usually constructed of 
copper, and because they readily burn out, are now made so that 
they may easily be replaced. Properly constructed sterilizers 
with removable bottoms may now be obtained and should be 
sought when purchasing, as they will save much annoyance and 
not a little expense. 

Sterilization by this method is accomplished in from a half 
to one hour at a temperature of from 150°-180° C. 

CHEMICAL STERILIZATION AND DISINFECTION. 

It is possible by means of certain chemical substances to 
destroy all bacteria and their spores, or by the same means to 
remove all their pathogenic properties; in other words, to dis- 
infect them. 

When it is desirable to use chemical disinfection in the 
laboratory, successful results may be obtained by employing a 
33- to 40-per-cent. solution of carbolic acid. Under ordinary 
circumstances this will accomplish the result in from twenty 
minutes to half an hour. It is, however, not reliable for the 
destruction of spores of resistant organisms, such as the spores 
of the anthrax bacillus. 

All materials and issues containing infectious organisms 
should be burned and all other cloths, test-tubes, flasksf dishes, 
etc., should be boiled in a 2-per-cent. soda (ordinary washing 
soda) for a half-hour, or should be exposed in the steam ster- 
ilizer for the same length of time. 



416 BACTERIOLOGIC METHODS. 

Intestinal evacuations are best disinfected with chlorinated 
lime, which should contain at least 0.25 per cent, of free chlo- 
rin. This solution should be mixed in equal parts with the 
material to be disinfected, and then should be allowed to stand 
for one or two hours before being disposed of. 

Sputum in which tubercle bacilli may be present, as well 
as vessels containing it, and the eating utensils of tuberculous 
patients, should be boiled with a 2-per-cent. soda solution for 
from a half to one hour, or should be exposed in the steam ster- 
ilizer for the same period. 



PREPARATION OF CULTURE MEDIA. 

Bouillon. — Eive hundred grams of freshly chopped, lean 
beef, free from fat and tendons, are soaked in a liter of water 
for twenty-four hours, during which time the temperature of the 
mixture is kept low by surrounding it with ice. 

At the expiration of this time the mixture should be strained 
through coarse muslin until a liter of fluid has been recovered. 
To this now add ten grams of dried peptone and five grams of 
common table salt. It is then to be rendered neutral or slightly 
alkaline by the addition of a few drops of a saturated sodium 
carbonate solution. The flask containing the mixture is then 
placed either in the steam sterilizer or upon a water-bath and 
kept at the boiling point until all the albumin has been coagu- 
lated, and the fluid portion is clear and of a pale-straw color. 
It is then filtered through a folded filter-paper and finally ster- 
ilized in the steam sterilizer by the fractional method (see page 
412). This is the original method of Koch which has been 
modified and improved in the following ways: — 

Neutralization. — Ordinarily this is accomplished hj the 
addition of a saturated solution of sodium carbonate, and the 
reaction determined by red and blue litmus paper. This 
sodium carbonate solution is not so good, however, as a strong 
solution of sodium or potassium hydroxid, because the carbonic 
acid arising during the process of neutralization with the sodium 
carbonate frequently produces a temporary acid reaction which 
later disappears on boiling. Exact titration with an 0.4-per-cent. 
solution of sodium hydrate, obviates this difficulty. The process 



PREPAEATION OF CULTURE MEDIA. 417 

is applied only after the bouillon has been deprived of all its 
coagulable albumin by boiling and has again been reduced to 
room-temperature. 

Technic. — First ascertain the exact volume of the fluid. 
From this sample take exactly 5 or 10 cubic centimeters and 
add a few drops of a 1-per-cent. alcoholic solution of phenol- 
phthalein as an indicator. The 0.4-per-cent. alkaline solution 
is placed in a graduated burette, and the solution to be tested 
in a porcelain dish or casserole. Now add the alkaline solution, 
drop by drop, until the bouillon turns a faint rose color. A 
second measured quantity of bouillon is treated in the same 
manner as a check, and if the amount of sodium hydrate solu- 
tion required to cause neutralization is the same or only slightly 
different, a simple calculation will indicate the amount of soda 
solution required to neutralize the bulk of the medium. Thus, 
if for 10 cubic centimeters of the bouillon would be required 1.5 
cubic centimeters of the 0.4-per-cent. solution of sodium 
hydrate, then for the remaining 980 cubic centimeters (original 
volume 500 cubic centimeters), it would require 98 times 1.5 
cubic centimeters of the soda solution to neutralize the total 
amount of bouillon, or 147 of the 0.4-per-cent. sodium hydrate 
solution would have to be added to the 980 cubic centimeters of 
bouillon to accomplish neutralization. 

To avoid over-diluting the bouillon by the weak alkaline 
solution, it is better to employ for this purpose a 4.0-per-cent. 
solution of NaOH, of which, only 14.7 cubic centimeters would 
be required. 

Not infrequently the filtered neutralized and sterilized 
bouillon will be found to contain a fine flocculent precipitate. 
This may be due either to an excess of alkalinity or to incom- 
plete precipitation of the albumin. The former may be corrected 
by the addition of a little dilute acetic or hydrochloric acid, fol- 
lowed by a second boiling, filtering, and sterilization. If due to 
imperfect precipitation of the albumin this may be corrected by 
reboiling and filtering. 

2. The substitution of prepared meat extract for the fresh 
extract is now almost universal. Any good stock meat extract 
will answer the purpose, and should be used in the strength of 
from two to four grams to the liter of water. Peptone and 

27 



418 BACTERIOLOGIC METHODS. 

sodmm chlorid are added, as in the original method of prepara- 
tion. 

The advantages of the meat extract are a decided shorten- 
ing of the time required for preparation of the media, and the 
production of a more uniform material. 

Nutrient Gelatin. — ^In making nutrient gelatin the bouil- 
lon is prepared first as outlined above, except that the reaction 
is corrected only after the gelatin has been completely dissolved. 
The reaction of the gelatin of the shops is frequently quite acid, 
entailing the addition of considerable more alkaline solution 
than required for the bouillon alone. 

The gelatin is added in sufficient quantity to make a 10- 
or 12-per-cent. solution. Its complete solution is accomplished 
either on a water-bath or over the bare flame. In the latter 
instance it must be constantly stirred to prevent burning the 
gelatin on the bottom of the pan. When the gelatin is com- 
pletely dissolved it can readily be filtered through an ordinary 
folded filter paper in a glass funnel. It not infrequently happens 
that in spite of the most careful technic the filtered gelatin is 
not perfectly clear; then clarification is required. For this pur- 
pose the mass must be redissolved, and when the temperature is 
between 60° and 70° C. the white of an egg, which has been 
beaten up with about 50 cubic centimeters of water, is added 
and thoroughly mixed in. This mixture is then again brought 
to the boiling point until coagulation of the egg albumin occurs. 
This process results in large, flaky coagula of albumin, which it 
is best not to break up, as fine flakes of albumin will clog the 
filter-paper and materially interfere with the process of filtra- 
tion. 

The filter-paper should always be moistened before filtering. 
If this is not done, the pores of the filter-paper will become 
clogged with gelatin and coagulated albumin, which will greatly 
interfere with rapid filtering. 

Gelatin should not, as a rule, be boiled for more than fifteen 
minutes, nor left in the steam sterilizer for more than thirty 
minutes, otherwise its property of solidifying will be impaired. 

As soon as the preparation of the gelatin is completed it 
should be sterilized in the steam sterilizer for fifteen minutes on 



NUTRIENT AGAR-AGAR. 4I9 

three consecutive days. The mouths of the containing flask or 
test-tubes should be completely plugged with raw cotton. 

Nutrient Ag^ar-Agar. — The preparation of this is difficult 
and tedious, and frequently fails from lack of patience or of 
experience, or both. Every worker has some slight modification 
of his own, but if, according to Abbott, the following directions 
are carefully carried out, the product will usually be satis- 
factory. 

Prepare bouillon in the usual way. As agar-agar re- 
acts neutral or faintly alkaline, the neutralization of the bouil- 
lon may be accomplished before the agar-agar is added. Finely 
chopped or powdered agar-agar is added to the bouillon in the 
proportion of 1 to 1.5 per cent. The mixture is then placed in 
an agate or earthen-ware boiler, and the height of the fluid, 
before boiling, marked upon its inside. If a liter of the 
medium is being made add about 275 cubic centimeters or more 
of water, and boil slowly for about two hours or until the excess 
of water that was added has been evaporated. Do not allow the 
fluid in the vessel to fall below the original level. If this occurs 
water must be added to bring the final amount up to one liter. 
At the expiration of two hours remove from the fire and cool 
rapidly by immersion in a pan of cold water. Stir constantly 
until the temperature of the mass has fallen to about 70° C, 
then add the white of one egg that has been previously beaten 
up in about 50 cubic centimeters of water. Mix this in well and 
allow to boil for half an hour longer, keeping the fluid up to the 
original one liter mark. The fluid is now easily filtered at 
room-temperature through a heavy folded filter. If properly 
prepared and filtered through a properly folded filter-paper, it 
should pass through at the rate of one liter in from fifteen to 
twenty minutes. 

Glycerin Agar-Agar.— The nutrient properties of agar- 
agar for certain organisms is greatly increased by the addition 
of 5-per-cent. glycerin. If glycerin is added to the agar-agar, 
it should be done after filtration and before sterilization. 

If after filtration the medium is found to contain flocculi, 
investigate the reaction. If it is quite alkaline it must be neu- 
tralized, boiled, and filtered again. If the reaction is neutral 



420 BACTERIOLOGIC METHODS. 

or only faintly acid, dissolve and again clarify with egg albumin 
as directed. All media must be neutral or only faintly alkaline 
to litmus, as but few organisms develop well on acid media. 

Dextrose and Lactose Litmus Agar. — One per cent, of dex- 
trose or of lactose may be added to hot sugar-free agar broth be- 
fore sterilization. Before using, sufficient of a 1 per cent, solu- 
tion of sterilized litmus is added to produce a 5 to 8 per cent, 
solution of the same in the culture medium. This addition 
should be tested for sterility before use. 

Blood-serum Media. — For small quantities of blood-serum 
for culture media purposes, blood may be obtained from 
small animals under such aseptic precautions as will guard 
against gross contamination. For laboratory purposes, where 
large amounts are required, it is best obtained from the slaughter 
houses. Under these conditions a certain amount of contamina- 
tion is unavoidable, though its extent may be limited by observ- 
ing certain precautions. 

Koch's original method, with slight variations, follows: — 

The animal from which the blood is to be obtained should 
be suspended by the hind legs so that its head is a few feet from 
the floor. The head should be held back when, with one sweep 
of a sharp knife, the throat is completely cut through. The 
blood as it spurts from the vessels should be collected in large 
glass jars which have been previously sterilized and dried with 
alcohol and ether. The jars should be provided with close- 
fitting cover and clamps capable of hermetically sealing them 
(large museum specimen jars will be found very satisfactory for 
this purpose). From two-gallon jars of blood there is usually 
recovered from 500 to 700 cubic centimeters of clear serum. 

The jars having been filled with blood, their covers are re- 
placed loosely and then they are allowed to stand quietly for 
about twenty minutes until clotting has begun. At the expira- 
tion of this time a clean glass rod is passed about the edges 
of the surface of the forming clot to break up any adhesions 
that have formed to the sides of the jar. The covers are now 
replaced and clamped down tightly, then with as little agitation 
as possible the jars are transferred to an ice chest, 40° F., where 
they should remain for from twenty-four to forty-eight hours. 
When the jars are removed from the ice chest a firm clot 



PREPARATION OF CULTURE MEDIA. 421 

will be found in the bottom of the jars. The serum is drawn 
off with a sterile pipette or syphon, and transferred to sterile 
glass cylinders. These cylinders are now placed on ice for an- 
other twenty-four hours, when the corpuscles will have settled 
to the bottom, leaving the serum above quite clear. This is then 
ready to be pipetted off into sterile test-tubes, about 8 cubic cen- 
timeters in each tube, or into flasks of 100 cubic centimeters' 
capacity. It is now ready for sterilization. This is accomplished 
by the intermittent method at a temperature of 70° C. for a 
period of one hour on five consecutive days. During the intervals 
it should be kept at room-temperature. After sterilization the 
tubes may be allowed to remain fluid or they may be coagulated 
by a short exposure to 80° C. For solidifying, the tubes should 
be placed in an inclined position in order to secure the greatest 
possible surface from the quantity of serum employed. 

The process of solidification requires constant attention if 
good results are to be obtained. No rule can be laid down for 
the time required to accomplish it, as this is not constant. Too 
high temperature and too rapid solidification results in an opaque 
and inelastic medium. 

When solidification is complete the tubes may be retained 
in a vertical position, and unless for immediate use must be 
protected from drying. This may be done by burning off' the 
superfluous ends of cotton and covering them with sterile rubber 
caps; or what is just as satisfactory and far cheaper, sterilized 
corks may be pushed down upon the cotton plugs. 

Owing to the employment of large quantities of serum, 
principally for the detection of diphtheria, the tedious method 
of Koch has been largely superseded by a number of more 
rapid modifications. 

Method of Councilman and Mallory. — By this method the 
serum is more quickly prepared. Rigid precautions against con- 
tamination of the blood during collection are not necessary, and 
the resulting medium, while neither transparent or translucent, 
fully meets the ordinary requirements of bacteriology. 

By this method the serum is decanted directly into sterile 
test-tubes as soon as obtained; it is then firmly coagulated in 
the slant position by exposure in a dry-air sterilizer at from 80° 
to 90° C. It is then immediately sterilized in the steam sterilizer 



422 BACTERIOLOGIC METHODS. 

at 100° C. for fifteen minutes on tliree consecutive days, as is 
the case with other media. 

Loeffler's Blood-Serum Mixture. — This mixture consists of 
one part neutral meat-infusion bouillon, containing 1 per cent, 
of grape-sugar and three parts blood-serum. The mixture is 
placed in test-tubes, sterilized, and solidified in exactly the same 
manner as described under blood-serum preparation, except that 
it requires a longer time and a higher temperature for coagula- 
tion. 

PREPARATION OF TUBES, FLASKS, ETC., FOR 
CULTURE MEDIA. 

While the media are in the course of preparation it is well 
to get the tubes, flasks, pipettes, etc., ready for their reception. 
These must be absolutely sterile. To this end both old and new 
tubes should first be boiled for a half-hour in a 2-per-cent. soda 
solution, then carefully swabbed out with appropriate bristle 
brushes. After rinsing in clear water they are immersed in a 
1-per-cent. solution of hydrochloric acid for a few minutes, then 
rinsed again and stood, round end up, to drain. When dry they 
are plugged with raw cotton carefully inserted so that there are 
no cracks or openings in the occluding cap. The plug should fit 
neither too loosely nor too tightly, but should fit firm enough to 
hold the weight of the tube when lifted by the protruding cotton. 

The tubes thus plugged are then placed upright in wire 
baskets and heated for one hour in the hot-air sterilizer to a 
temperature of 150° C. Tubes so prepared, if undisturbed, will 
remain sterile for an indefinite period. 

Filling Tubes. — The tubes are best filled with the aid of 
a separating funnel, though if not convenient the tubes can, 
with a little care, be successfully filled directly from the flasks. 
It is not necessary to sterilize the funnel as the media in the 
tubes is to be sterilized as soon as they are filled. In any case, 
care should be observed to prevent any of the medium from 
coming in contact with the mouth of the tubes, which would 
cause the cotton plugs to adhere to it, making them hard to 
remove, presenting a very untidy appearance and materially in- 
terfering with subsequent manipulations. 

After filling, the tubes are ready for final sterilization. This 



TECHNIC FOR PLATES AND PETRI DISHES. 423 

is accomplished in the steam sterilizer by the three-day frac- 
tional method. 

TECHNIC FOR PLATES AND PETRI DISHES. 

Plates. — The plate method can be employed with both agar 
and gelatin, bnt cannot be practiced with blood-serum, because 
the latter when once it is solidified cannot again be rendered 
liquid. 

Plates are usually referred to as a set. This term includes 
three separate plates each representing a mixture of the organ- 
ism in a state of greater dilution. The plates are numbered 
1, 2, and 3. A set of plates may be prepared as follows : Three 
tubes, each containing the requisite amount of gelatin or agar- 
agar, are placed in a water-bath and warmed until the medium 
is fluid. Agar-agar becomes fluid at about the temperature of 
boiling water; gelatin is fluid between 35° and 40° C. In the 
case of the agar-agar tubes, after liquefying they must be 
cooled to 40° C, at which temperature they remain fluid while 
the organisms are introduced. If this cooling is omitted the tem- 
perature of the medium when the organisms are inoculated will 
be sufficiently high to destroy their vitality. 

The medium now being liquid and of a proper temperature, 
the material containing the organisms is taken up on a sterile 
platinum wire-loop and transferred to tube 1, where it is thor- 
oughly disintegrated and mixed by rubbing against the side of 
the tube. The more carefully this is done the more uniform will 
be the distribution of the organisms and the better the final re- 
sults. The loop is now again sterilized by passing through the 
flame, and when cool three loops full from tube 1 are trans- 
ferred to tube 2, where they are carefully stirred in. Again, 
the wire is sterilized and the same manipulation carried out be- 
tween tubes 2 and 3. This completes the dilution. 

During these manipulations, which must be done rapidly if 
agar is employed, the temperature of the water bath must be 
kept between 39° and 43° C. If the temperature falls below 
38° C. the agar-agar will become solidified and can then only 
be reliquefied by the application of heat sufficient to destroy the 
organisms introduced. 

After inoculation the contents of these tubes are poured out 



424 BACTERIOLOGIC METHODS. 

upon the sterilized plates, cooled, and incubated for twenty-four 
or forty-eight hours. 

Petri Dish Method. — This process materially simplifies the 
original technic of the plate method. It consists in substituting 
for the flat plates of glass small, round double-glass dishes having 
about the same surface area as the original plates. The inocu- 
lated and liquid media is poured directly into these, their covers 
are immediately replaced, and they are set aside to cool. In all 
other respects the process is the same as Koch's original plate 
method. These dishes have vertical sides which prevents over- 
flowing of the medium. A convenient size for this method meas- 
ures about 12 centimeters in diameter, but dishes of other 
sizes are easily obtainable. The dishes are readily sterilizable 
by hot air or steam, and have the great advantages that the 
danger of contamination is reduced to a minimum, since after 
sterilizing the plates do not have to be separated until the pour- 
ing on of the medium, and then only for a moment. 

THE INCUBATOR. 

After the plates have been made and solidifled they should 
be transferred to an incubator where a uniform and favorable 
temperature may be maintained. Various types of incubators 
have been devised, but since the principle and purpose of all is 
the same, a general description of their construction and method 
of employment is all that is required. 

The incubator or thermostat (Fig. 76) consists essentially 
of a copper chamber of convenient size provided with double 
walls, between which heated water circulates. The incubator 
chamber has a close-fitting door of heat-proof construction, and 
usually within this is a second door provided with a glass front, 
which permits inspection of the interior of the incubator with- 
out actually opening the chamber, and so reducing the tem- 
perature. The whole apparatus is set upon an enclosed base 
and is covered with asbestos board to prevent loss of heat from 
radiation. In the top of the chamber is a small opening fitted 
with a perforated cork through which a thermometer projects 
into the interior. Two other openings are provided, one for the 
thermometer which records the temperature of the circulating 
water, the other for the thermo-regulator. At one side of the 



THERMOSTATS AND INCUBATORS. 



425 



apparatus is a vertical water gauge provided with an upper 
opening for the introduction of water, and a stop-cock below 
for drawing off the water when occasion required. 

When in operation the apparatus should be kept full of 
water; otherwise^ the object of the water jacket will be defeated 
and the temperature of the interior of the chamber will not be 
maintained. Heat is supplied to the incubator by a gas burner 





Fig. 76.— Thermostat or Incubator. (A. H. T. Co.) 

placed within the inclosed space below the chamber. The par- 
ticular form of burner usually employed is known as "Koch's 
safety burner/' which is so constructed that should by accident 
the light be extinguished, the flow of gas would be almost im- 
mediately shut oif. An ordinary Bunsen burner, well protected 
from sudden gusts of air, will serve the purpose equally as well. 
The Thermo-Eeg^ulator.— The efficiency of the thermostat 
depends upon the proper and uniform temperature which is 
maintained by the thermo-regulator. A satisfactory regulator 
should permit of a fluctuation of not more than 0.3° C. in the 
temperature within the chamber of the apparatus. 



426 BACTERIOLOGIC METHODS. 

The commonest form of regulator is constructed upon the 
principle involving the expansion and contraction of fluids under 
the influence of heat and cold. By means of such expansion and 
contraction the amount of gas passing from the source of sup- 
ply to the burner is modified as the temperature of the water in 
the incubator rises or falls. 

For the successful employment of clinical laboratory meth- 
ods, the thermo-regulator is useful, but not essential. In a room 
of fairly uniform temperature the flow of gas can be regulated 
by hand until the internal temperature of the incubator is 37° 
C. After this, if the water level is maintained and the apparatus 
protected from protracted change in the surrounding tempera- 
ture, the variation in the internal temperature will be so slight 
that it need not be considered. 

DESCRIPTION OF COMMON DISEASE-PRODUCING 
ORGANISMS. 

The Tubercle Bacillus. — This organism has been fully dis- 
cussed in Chapter II, on Sputum, as it is in this material that 
search is usually made for purposes of diagnosis (see pages 31 
to 39). The tubercle bacillus may be found in any secretion or 
excretion as well as in any organ of the body. In different 
localities the details of its isolation and detection must naturally 
differ, although after the preliminary technic of isolation and 
specimen preparation the methods of fixation, staining, and 
microscopic search are similar to those already described else- 
where, and will not be repeated here. 

Tuberculosis of tissues other than the lungs is probably 
best demonstrated by inoculation experiments carried on in 
guinea-pigs, and even in sputum examination, supplementary 
animal inoculation is desirable, when available, as a means of 
control. This seems almost essential to-day when early diagno- 
sis is insisted upon, because investigators have shown that stain- 
ing methods fail to give positive findings in doubtful cases, while 
properly conducted inoculations almost never fail. 

One of the unsettled controversies which has been waged 
among bacteriologists and pathologists during the past six years 
is that of the diagnostic value of finding acid-fast bacilli in the 
blood. In spite of the time that has elapsed since the statement 



COMMON DISEASE-PRODUCING ORGANISMS. 427 

of Eosenberger, the question of the prevalence of tubercle bacilli 
in the circulating blood of all cases of tuberculosis is still un- 
settled, the preponderance of evidence pointing to the belief that 
tuberculosis is not regularly, although it occasionally may become, 
a bacteremia. 

Through the courtesy of Dr. Eandle C. Eosenberger,i the 
originator of the test, I am enabled to give in full the methods 
employed by him in isolating these acid-fast organisms from the 
blood and feces. 

The Method. — The Blood: Take 5 or 10 cubic centi- 
meters of blood from a vein in the arm of the patient (after 
having cleansed the part thoroughly) . The blood is best drawn 
through a large hypodermic needle fitted with a short piece of 
rubber tubing, which is held so as to extend into a sterile test- 
tube or centrifuge-tube. The tube should contain 5 cubic 
centimeters of a sterile 2 per cent, solution of neutral sodium 
citrate to prevent the least coagulation. Antiformin is now 
added (pure) until the blood is entirely destroyed. Only a very 
small quantity of antiformin is needed and should be added, a 
few drops at a time. The specimen is now centrifugated for 
twenty minutes, the supernatant fluid carefully poured off and 
the small precipitate carefully washed with sterile distilled 
water to remove the antiformin; again centrifugate, collect 
the precipitate on a perfectly clean slide, dry, fix, and stain for 
five or ten minutes with cold carbol-fuchsin ; wash in water and 
then apply Pappenheim's solution. The slide is left in Pappen- 
heim's solution for two or three minutes, washed with water and 
the stain again applied for five minutes; this procedure is 
repeated until the slide actually receives twenty minutes' inter- 
mittent immersion in the Pappenheim solution. Wash in 
water, dry and mount, or examine immediately without mount- 
ing for red bacilli. 

The Feces. — Make a spread from any portion of the stool 
upon a clean slide; dry, fix and stain as above (for blood). If 
acid-fast organisms are not found by this method, take about 
5 grams of the specimen and add pure antiformin, 1 part 
antiformin to 4 or 5 parts feces, and allow this to act for 
from fifteen to twenty minutes ; then add sterile distilled water : 

1 Personal communication. 



428 BACTERIOLOGIC METHODS. 

centrif ugate ; wash the sediment with distilled water; again 
centrif ugate ; collect the sediment npon a clean slide, and spread 
and stain as before. As a rule, by this procedure all other 
bacteria are destro3^ed, so that the only organisms found will be 
those of tuberculosis. 

BACILLUS OF DIPHTHERIA. 

From the grayish-white deposit on the fauces of a diph- 
theritic patient, prepare a series of cultures in the following 
way:— 

Have prepared a few tubes of Loffler's blood-serum. Pass 
a stout platinum needle which has previously been sterilized into 
the membrane and rub it around there; then being careful that 
it touches nothing else, rub it carefully over the surface of two 
tubes. The tubes are then immediately replugged and placed in 
the incubator. If the case be one of true diphtheria, the tubes 
will be ready for examination the following day. 

The blood-serum mixture is to be preferred to the ordinary^ 
plate method because the organism of diphtheria grows better 
on this medium than upon any other; it is also a differential 
method in a general sense, because other organisms do not grow 
well on Loffler's serum; hence a luxuriant growth at the expira- 
tion of twenty-four hours should always be considered diph- 
theritic until proven otherwise. 

Appearance. — After twenty-four hours the tubes present a 
characteristic appearance. Their surfaces are marked by more 
or less irregular patches of white or cream-colored growths, 
which are usually more dense at the center than at the periphery. 

Staining. — From this culture smears are made upon clean 
cover-slips or slides, dried and fixed in the usual wa}^ and then 
stained with Loffler's alkaline meth3dene-blue. There will now 
be seen, in a typical case, upon microscopic examination, slightly 
curved bacilli of irregular size and outline. In some cases they 
will be more or less clubbed at one or both ends; sometimes 
they are spindle shaped or may present curved edges. They are 
rarely or never regular in outline. Many of these irregular rods 
are seen to be marked at circumscribed points in which their 
protoplasm is deeply stained. This irregularity in outline and 



BACILLUS OF DIPHTHERLi. 429 

appearance is the morphologic characteristic of the bacillus of 
diphtheria. 

THE GONOCOCCUS OF NEISSER^ 

On microscopic examination, the pus from an acute case of 
gonorrhea will show numerous small bodies, usually arranged 
in pairs. These cells will be found both within and without the 
protoplasm of the pus-cells. The cells containing the gonococcus 
are usually crowded with the organisms, though the majority 
of pus-cells do not contain them. This organism, on account of 
its frequent arrangement in pairs, is often called the diplococcus 
of gonorrhea. It is always found in gonorrheal pus and often 
persists into the convalescent stage after the external discharge 
has disappeared. It is easily detected in the pus of acute in- 
vasion, while in the subacute and chronic conditions its detec- 
tion is often a matter of considerable difficulty. 

Cultivation. — It does not grow upon the ordinary culture 
media, and can only be isolated in culture through the employ- 
ment of special methods. Blood or blood-serum is a necessary 
constituent of all media for the artificial cultivation of this 
organism. Some investigators have been successful in growing 
it upon other body fluids, such as ascitic fluid, pleural effusion, 
and the fluid from ovarian cysts. A useful medium may be 
prepared by mixing equal parts of human blood-serum with ordi- 
nary sterilized nutrient agar-agar. This is accomplished by 
liquefying the agar, maintaining it at a temperature of 50° C. 
until after the mixture is made, after which it is allowed to 
solidify. 

Distinguishing Characteristics. — 1. It is seen practically 
always in the form of a diplococcus, having the characteristic 
biscuit form with the long diameters of the individual cells 
apposed. 

2. In gonorrheal pus some organisms are practically always 
found within the protoplasm of some of the cells. 

3. It stains readily with the ordinary staining reagents, but 
it is promptly decolorized by Gram's. 

4. It fails to develop on the ordinary artificial media (sepa- 
rating it from the Diplococcus intracellularis meningitidis, which 
grows freely). 



430 BACTERIOLOGIC METHODS. 

5. It has no pathogenic properties for the lower animals. 

Staining. — The ordinary stains are satisfactory, one of 
the simplest being a 1 or 2 per cent, aqueons solution of methy- 
lene-blue, which gives a very clear picture. 

Most important as a differential method is its failure to 
retain its color when treated by Gram's method. 

MENINGOCOCCUS (MICROCOCCUS MENINGITIDIS). 

This organism was extensively studied first by Weichsel- 
baum in 1887, who described its relation to acute inflammatory 
involvement of the meninges. It is now recognized as the 
specific cause of epidemic cerebrospinal meningitis. 

Morphology. — In cover-slip preparations the meningococcus 
appears as a diplococcus or in tetrad form. Characteristically 
it appears within the polymorphonuclear, these white cells often 
being so densely packed with the organism as to obscure the 
iiucleus. They are readily decolorized by Gram's. This charac- 
teristic serves to distinguish them from the ordinary streptococci 
and from the Diplococcus pneumonice. They do not possess a 
capsule, but involution forms are common (Jordan). 

Culture Characteristics. — They grow with difficulty on the 
usual culture media and are best cultivated on Loffler's blood- 
serum. They are of poor vitality and, unless immediately 
planted, efforts at culture may be unsuccessful because of the 
death of the organisms. 

Staining Methods. — The cover-slip after proper fixation 
may be stained with alkaline methylene-blue or any other ordi- 
nary stain, and for differentiation the Gram method should be 
employed. 

TYPHOID BACILLUS. 

This organism has been known since 1880 and is now 
recognized as the specific cause of typhoid fever in at least 
the great majority of cases, so designated. It must be remem- 
bered that cases clinically diagnosed as typhoid fever, especially 
if they vary in some details from the classical picture of this 
disease, may be due to one of the paratyphoid organisms. This 
will account for the failure to obtain a specific agglutination, 



TYPHOID BACILLUS. 431 

and other laboratory examinations for the demonstration of this 
disease. 

Morphology and Staining. — The typhoid bacilli are short, 
rather thick rods, having rounded ends, often growing in long 
threads. They differ from the other members of the colon 
group in that they are more slender and are usually longer. 
These organisms take the ordinary aniline stains, but less in- 
tensely than the majority of organisms. Bipolar staining is 
sometimes seen, and like the colon and paratyphoid group they 
fail to retain the color when treated with Gramas. The organ- 
isms usually possess numerous fiagella, which may spring from 
the sides as well as from the ends of the rods. Short rods may 
be encountered, which possess but a single terminal fiagellum. 
The organism is, therefore, motile, aerobic, and a facultative 
anaerobe. The organism does not form spores. 

The motility of the organism facilitates the demonstration 
of the Widal agglutination reaction discussed in the chapter on 
Serodiagnosis (see page 438) . 

The organisms are usually present in the blood in patients 
ill with typhoid fever. This fact is of value in early diagnosis, 
especially when the Widal is negative, although it must be re- 
membered that McFarland has examined the blood of typhoid 
patients daily throughout the course of the disease and found 
10 per cent, of cases always negative. 

Blood Cultures. — The following method may be employed 
for obtaining blood cultures in typhoid fever: A 10 cubic centi- 
meter Leur syringe is sterilized and protected in a large glass 
tube. The patient^s arm is constricted above the elbow by means 
of a rubber bag of a sphygmomanometer, until the veins at the 
bend of the elbow stand out prominently. The space at this 
point is thoroughly scrubbed with alcohol and painted with tinc- 
ture of iodin. From 8 to 10 cubic centimeters of blood are then 
drawn up into the syringe; 1 to 1.5 cubic centimeters are mixed 
well with about 100 cubic centimeters of sterile broth in a flask ; 
1 to 1.5 cubic centimeters with about 8 cubic centimeters of 
sterile bile, and about 1 cubic centimeter is added to each of 
3 agar plates. All of these are then incubated for twenty- 
four hours. If, at the end of that time, hanging drops show 
motile bacilli resembling typhoid organisms in their morphology 



432 BACTERIOLOGIC ISiETHODS. 

and manner of motion, snbcnltures are made on litmus milk, 
glucose agar, agar slant, broth, and peptone. It is advisable to 
subculture at the end of twenty-four hours, whether the hanging 
drops show motile bacilli or not, in which case the original 
cultures should be incubated for another twenty-four hours 
before subculturing. In most cases the earlier subculturing will 
hasten the diagnosis. A growth of typhoid organisms on the 
original blood-agar plates is the most difficult to obtain, but 
even then it may be the means of ruling out streptococcus and 
pneumococcus infections. If the subcultures show a character- 
istic growth in the agar slant, with no acidification of the litmus 
milk, no gas in the glucose agar, no indol in the peptone culture, 
and a characteristic motile bacillus in the broth, the diagnosis 
may be considered sufficiently established (Jordan). 

Elimination of the Typhoid Bacillus Fkom the Body. 
— The typhoid bacillus frequently gets into the secretions and is 
at some time during the disease present in the urine in about 20 
per cent, of cases. It is occasionally found in the sputum, is 
almost always present in the gall-bladder, and is always found in 
the feces. The bacillus usually disappears from the body at about 
the end of the fifth week, but may occasionally remain for 
months in the urine and throughout life in the gall-bladder. 
Examinations of typhoid fever cases show that from 1 to 5 per 
cent, continue to pass typhoid bacilli for many months or even 
years. This knowledge has given rise to the recognition of a 
special class of individuals known as typhoid carriers. The 
majority of such persons are women. 

CLASSIFICATION OF BACTERIA ACCORDING TO 
STAINING PROPERTIES. 

Bacteria may be divided into two groups, according to 
whether they are positive or negative to G-ram. By positive 
we mean that the bacteria retain the primary color or the 
gentian violet after decolorization with iodin. The term 
"negative to Gram" indicates that the bacteria lose the primary 
color after treatment with alcohol and take up the counterstain, 
such as Bismarck brown or carmin. 

Gram's Method. — Objects are first treated with an anilin- 
water solution of gentian-violet which is made after the formula 



CLASSIFICATION OF BACTERIA. 433 

of Koch-Ehrlich (see Appendix for preparation of stain). After 
t-taining in this solution for fifteen to thirty minutes the excess 
of stain is drained off and the film treated for five minutes with 
G-ram^s iodin solution (see Appendix). They are next trans- 
ferred to alcohol and thoroughly rinsed. If careful examina- 
tion at this point reveals any violet color in the film^ it must 
again be treated with the iodin solution until all violet color is 
removed. After a final washing in alcohol the specimen may 
be mounted and examined or a counter-stain of carmin may 
first be employed. 

"Wright's Modification. — Stain for one minute in carbol- 
gentian- violet (see Appendix). Wash in water from thirty 
to sixty seconds. LugoFs solution is then allowed to act upon 
the specimen for one to three minutes. Wash and dry. Dif- 
ferentiate with anilin-xylol (2:1) to which 1.5 per cent, of 
acetone has been added, for one or two minutes. Wash with 
xylol, dry, and counter-stain with dilute carbol-fuchsin (1: 10) 
for about one minute, during which the specimen should be 
warmed slightly. The specimen is finally washed, dried, and 
mounted for examination. 

The process of decolorizing is only a relative one, some 
bacteria decolorizing more readily than others, so that much 
depends upon the intensity of the decolorizing reagent, and also 
upon the time during which it is allowed to act. The counter- 
stain method with dilute carbol-fuchsin is a differentiated proc- 
ess indicating those organisms that have been decolorized. All 
decolorized organisms by this method take on a red color. This 
counter-stain is of particular value where pictures of a number 
of bacteria are made, as in sputum examination. 

The following organisms retain the violet stain by Gram's 
methods : — 

Streptococci. 

Staphylococci. 

Bacillus tuberculosis. 

Bacillus anthracis. 

Bacillus aerogenes capsulatus. 

Bacillus diphtherias. 

Diplococcus pneumonias (Frankel's). 

Bacillus tetanus. 

28 



434 BACTERIOLOGIC METHODS. 

Smegma bacillus. 

Lepra bacillus. 

Timothy bacillus. 

Saprophytic cocci of the urethra. 

The following organisms are decolorized by Gram's 
method : — 

Diplococcus meningitidis intracellularis. 

Bacillus pyocyaneus. 

Micrococcus gonorrhoeae (Neisser's). 

Bacillus malignant edema. 

Bacillus influenzae. 

Bacillus typhosus. 

Bacillus choleras. 

Bacillus pneumoniae (Friedlander's). 

Morax-Axenfeld. 

Colon bacillus. 

Bacillus pestis. 

Micrococcus catarrhalis. 

Koch- Weeks bacillus. 

Bordet-Gengou bacillus. 

The bacillus of Friedlander, the diphtheria bacillus, and 
the Diplococcus intracellularis are somewhat variable in their 
behavior toward Gram's stain, and may or may not decolorize, 
but the most usual type is taken in the column of the classifica- 
tion table. 

LOFFLER'S METHOD OF STAINING FLAGELLA. 

It is essential that the bacteria be evenly and not too nu- 
merously distributed over the cover-slip. 

Preparation of Cover-slip. — ^The glasses must be perfectly 
clean. Lay six cover-slips on an even surface, and place in the 
center of each a small drop of distilled water. From the mate- 
rial to be examined, transfer a minute quantity to the drop of 
water on the first cover-slip; from this one transfer a minute 
quantity to the second, and so on to the sixth. This insures a 
varying dilution of the organisms in the different preparation. 
They are then all spread, dried, and fixed in the usual way. 
The cover-slip preparations are next warmed in Loffler's mor- 
dant (see Appendix). 



LOFFLER'S METHOD OF STAINING FLAGELLA. 435 

A few drops of this solution is placed upon the preparation, 
which is held over a low Bunsen flame until it begins to steam. 
It should not be boiled. After steaming for a few moments the 
mordant is washed ofl with water and then with alcohol. The 
bacteria are now stained in the Koch-Ehrlich anilin-water- 
fuchsin solution (see Appendix). 

When treated in this way various bacteria behave differ- 
ently, the flagella of some staining readily; others require the 
addition of an alkali in varying proportion to obtain the best 
results; others again stain best after the addition of an acid. 

To meet these conditions an exact 1 per cent, solution of 
caustic soda in water must be prepared, and also a solution of 
sulphuric acid of such strength that 1 cubic centimeter will 
exactly neutralize 1 cubic centimeter of the alkaline solution. 

For the different bacteria which have been studied by this 
method Loffler recommends that one or the other of these solu- 
tions be added to the mordant before using, in the following 
proportions : — 

Of the Acid Solution. 

For Spirillum concentricum, no addition of either acid or alkali. 

For Spirillum cholera Asiaticse, % to 1 drop to 16 cubic centi- 
meters of the mordant. 

For Spirillum Metchnicovi, 4 drops of acid to 16 cubic centimeters 
of the mordant. 

For Spirillum rubrum, 6 drops of acid to 16 cubic centimeters of 
the mordant. 

For Bacillus pyocyaneus, 5 drops of acid to 16 cubic centimeters 
of the mordant. 

Op the Alkaline Solution. 

For Bacillus mesentericus vulgaris, 4 drops of alkali to 16 cubic 
centimeters of mordant. 

For Bacilllus Micrococcus agilis, 20 drops of alkali to 16 cubic 
centimeters of mordant. 

For Bacillus typhosus, 22 drops of alkali to 16 cubic centimeters of 
mordant. 

For Bacillus subtilis, 28 to 30 drops of alkali to 16 cubic centi- 
meters of mordant. 

For Bacillus malignant edemae, 36 to 37 drops of alkali to 16 
cubic centimeters of mordant. 

For Bacillus symptomatic anthrax, 35 drops of alkali to 16 cubic 
centimeters of mordant. 

The drops used run 22 to the cubic millimeter. 



436 BACTERIOLOGIC METHODS. 

CAPSULE STAINS. 

Welch's Methods :— 

1. Cover the film with glacial acetic acid. 

2. Draw off acetic acid and treat the film several times with 
anilin-gentian-violet. 

3. Wash in 0.85 per cent. NaCl solution and examine in 
the same solution. Avoid the use of water at any stage. The 
capsule appears as a pale violet halo around the deeply stained 
bacterium. 

Muir's Method : — 

1. Prepare, dry, and fix film in the ordinary manner. 

2. Flood the film with carbol-f uchsin ; warm until steam 
begins to rise ; allow the stain to act for thirty seconds. 

3. Wash quickly with methyl alcohol. 

4. Wash thoroughly with water. 

5. Then apply Muir^s mordant (see Appendix, page 488) 
for five seconds: — 

6. Wash off quickly and thoroughly with water. 

7. Treat with methyl alcohol for about sixty seconds (the 
preparation should now be pale red) . 

8. Wash thoroughly in water. 

9. Counterstain with 1 per cent, aqueous methylene-blue 
solution for thirty seconds. 

10. Wash in water. 

11. Deh3'drate in alcohol. 

12. Clear in xylol and mount in xylol balsam. 

METHOD OF STAINING CAPSULATED 
BACTERIA IN BODY FLUIDS. 

Smith^ describes his method of staining capsulated bacteria 
in body fluids as follows: Make a thin smear from fresh 
sputum, lung, pleural, or pericardial exudate. Pass through 
flame. Cover with 10 per cent, aqueous solution of phospho- 
molybdic acid four to five seconds. Wash in water. If the 
micro-organism is Gram staining, like the pneumococcus or 
Streptococcus mucosus capsulatus, stain with anilin oil-gentian 
violet, steaming for from one-quarter to one-half minute. Wash 



2 Bun. Johns Hopkins Hosp., 1892. iii, p. 128. 

3 Boston Med. and Surg. Jour., Nov. 24, 1910. 



CAPSULE STAINS. 437 

in water. Treat with Gram's solution of iodin; steam for from 
one-qnarter to one-half minute. Decolorize with 95 per cent, 
alcohol. Wash in water. Stain with 6 per cent, aqueous solution 
of eosin, for from one-half to one minute, warming gently. Wash 
in water. Wash in absolute alcohol. Clear in xylol and mount 
in Canada balsam. The capsule will be found to be distinct, 
clear cut, eosin stained, about the Gram-stained micro-organism. 
If the micro-organism is Gram decolorized; after covering the 
smear with phosphomolybdic acid and washing, stain with 6 
per cent, aqueous solution of eosin, for from one-half to one 
minute, warming gently. Wash in water. Counter-stain with 
Loffler's methylene-blue, for from one-quarter to one-half 
minute, warming gently. Wash in absolute alcohol. Clear in 
xylol and mount in Canada balsam. The capsule will appear 
eosin stained about the blue-stained micro-organism. 

Hiss's Method.4 — 1. Smear the sputum in a very thin layer 
without the addition of water, dry and fix by heat. 

3. Cover the preparation with a mixture containing 5 cubic 
centimeters of a saturated alcoholic solution of gentian violet 
in 95 cubic centimeters of distilled water. Heat till steam be- 
gins to rise. 

3. Wash off the dye with a 20 per cent, solution of copper 
sulphate. 

4. Dry and mount in dammar. 

4 Wood's "Chemical and Microscopical Diagnosis," 2d ed., 1910. 



XIV. 

SERODIAGNOSIS. 



AGGLUTINATION REACTIONS. 

SPECIAL AND SPECIFIC REACTIONS. 

Agglutination. — We know that in the natural infections of 
man peculiar changes occur in the blood-serum due to the influ- 
ence of specific bacteria or their soluble toxins. Among these, 
the most familiar occurs in typhoid fever and is shown by the 
agglutination of these motile organisms in the Widal reaction. 

Definition. — Agglutination in a bacterial sense refers to 
the clumping or precipitation of micro-organisms by the action 
of serum. While first employed as the G-ruber-Widal reaction 
in the diagnosis of typhoid fever, subsequent investigations have 
demonstrated the applicability of this reaction in the blood of 
a number of specific infections. 

Normal serum may agglutinate many bacteria, as typhoid, 
colon, pyocyaneus, and dysentery, but not the streptococcus and 
many others. This agglutination only occurs in low dilutions, 
though the typhoid has been found to agglutinate in dilutions 
of 1 to 30. This point is to be remembered in practical diag- 
nosis, and care must be taken in the performance of these tests to 
carry them out with sufficient dilution to avoid a spurious reac- 
tion, due to a too concentrated serum. 

THE SPECIFIC TYPHOID OR WIDAL REACTION. 

There are two methods: 1. The macroscopic or naked- 
eye observation of the clumping and sedimentation of a homo- 
geneous suspension of bacteria in a test-tube. 2. The micro- 
scopic observation of the clumping of the organisms when mixed 
in diluted serum and mounted in a hanging-drop preparation. 

For the hanging drop it is necessary to have slides with 
concave depressions in the middle. A drop of the serum under 
(438) 



SPECIFIC TYPHOID OR WIDAL REACTION. 439 

examination is placed in the center of a cover-glass, which is 
then placed drop-side down over the depression, its edges being 
sealed by vaselin or paraffin. In this preparation with the 
aid of a microscope the loss of motility incident to agglutination 
is readily observed. 

Different typhoid cultures vary in their susceptibility to 
clumping, so that each observer should make himself familiar 
with the peculiarities of his own cultures. 

In order to have fresh typhoid cultures always at hand it is 
best to transfer glycerin-agar cultures every eight to fourteen 
days, or otherwise cultures degenerate and die off. A bouillon 
culture is prepared from the water of condensation of the agar 
culture twenty-four hours before the test is to be made and is in- 
cubated at room temperature. 

Microscopic Reaction. 1 — Five hanging-drop preparations 
should be made as follows : — 

(a) One loopful of bouillon culture and 1 loopful of pooled 
serum. This is the control (pooled serum is serum prepared 
from the blood of several individuals known to be free from 
typhoid fever). 

(h) One loopful of culture + 1 loopful undiluted specific 
serum = 50 per cent. 

(c) One loopful of culture + 1 loopful 10 per cent, 
serum (1 part serum diluted with 9 parts normal saline) = 5 
per cent. 

(d) One loopful culture + 1 loopful 1 per cent, serum (1 
part serum diluted with 99 parts normal saline) =0.5 per cent. 

(e) One loopful culture and 1 loopful 0.1 per cent, serum 
(1 part 1 per cent, serum diluted with 9 parts normal saline) = 
0.05 per cent. 

These are examined from time to time, a record being made 
of the time that the dilutions were made. The control should not 
agglutinate. The 5 per cent, dilution should remain non- 
agglutinated for thirty minutes before reporting a negative reac- 
tion. The 0.5 and 0.05 per cent, preparations may or may not 
agglutinate, depending on the potency of the serum. ^ 



1 Eyre : "Bacteriologic Technic," 2d Edition, 1913. 

2 The Thoma-Zeiss Hemocytometer tubes may be used for diluting the 
serum. 



440 SERODIAGNOSIS. 

Microscopic Reaction. — 1. Place from 90 to 100 cubic 

millimeters of diluted serum into each, of 3 sterile test-tubes: 
a = 10; & = 1 per cent., and c = 0.1 per cent, dilu- 
tions. In a fourth tube place the same amount of 50 per cent, 
pool serum. 

2. Add to each tube the same amount (90 or 100 cubic milli- 
meters) of twent3^-four hours' bouillon culture, stop tubes with 
cotton, and stand upright in an incubator at 37° C. for one to 
two hours. 

In a positive reaction the pool-serum tube should show no 
change, while the dilution tubes will show a granular deposit, 
which is easily distinguishable from a uniform turbidity shown 
in a negative reaction. 

A simple method that is practised by many boards of health 
and in private laboratories is to dry a few drops of patient's 
blood on a piece of paper and transmit that to the laboratory 
for examination. Here the laboratory worker becomes accus- 
tomed to estimate the amount of blood forming the dried drop, 
and this is diluted with an equal amount of normal saline. 
After thorough softening of the drop, the whole is transferred 
by means of the loop to a watch-glass or slide. From this 50 
per cent, dilution subsequent dilutions are made as described 
above. 

The Serum Dilution. — It is usually held that a dilution of 
1 to 40 or 1 to 50 with normal salt solution is sufficient 
to eliminate the possibility of false agglutination by normal sera, 
and at the same time sufficiently low to permit of the good 
reaction to nearly all typhoid cases, excepting possibly advanced 
convalescents. 

If agglutination occurs in this preparation, we note, with 
the aid of the high power, that, in the course of from fifteen 
minutes to an hour as the micro-organisms swim about, a few 
as they come in contact have a tendency to remain in this 
relation. In the course of a few more minutes other organisms 
join this group and other groups form throughout the field; 
motility becomes gradually less until it ceases entirely in the 
typical reaction. The complete change occurs in from six to 
eight hours. Not less than five bacteria must become permanently 
agglutinated to constitute a positive reaction. The test is most 



AGGLUTINATION REACTION. 44I 

decisive when large masses of permanently agglutinated organ- 
isms are formed which can be seen with the lower power. 

Method of Bass and Watkins. — These workers have in- 
troduced a modification of the macroscopic agglutination test, 
which is simple, reliable, and quick. It has the advantage that 
it can be carried out at the bedside, the result being known in 
two to three minutes. Little equipment is required. The sus- 
pension consists of 100 million killed typhoid organisms per 
cubic centimeter in 1.7 per cent. NaCl solution to which 1 per 
cent, formalin is added. 

The Test. — Allow a full drop of blood to fall into 4 drops 
of water and mix thoroughly. Instead of this, one may make 
a blood-smear (using approximately % <irop of blood and dis- 
solving this on the slide with 1 drop of water, the mixture being 
stirred with a tooth-pick or similar substance). With this 
diluted blood (1 to 4) mix an equal amount of the above sus- 
pension of typhoid bacilli on a glass slide. Tilt the slide from 
side to side so as to keep the mixture flowing back and forth. 
If the reaction is positive a grayish, mealy sediment appears 
within one minute, usually in less. This sediment appears 
in the fluid around the edges and tends to collect there. If the 
agitation is continued, the clumps increase in size for two to 
three minutes. If the reaction does not appear in this time, 
it will not appear at all. When the reaction is negative, no 
agglutination occurs and the mixture remains as clear and un- 
changed as when first put on the slide. 

AGGLUTINATION REACTIONS IN DISEASES 
OTHER THAN TYPHOID. 

Paratyphoid Infections. — A certain number of cases clin- 
ically resembling typhoid fever do not give an agglutination 
reaction with typhoid bacilli. From some of these cases a 
group of bacilli has been isolated, the members of which resemble 
typhoid bacilli in some of their cultural characteristics. They 
differ from the latter, however, especially in the formation of 
gas in glucose bouillon and by their serum reactions. The 
blood-serum of a patient suffering from infection with one of 
these varieties (paratyphoid) will agglutinate the species isolated 
from the patient's blood in the high dilutions; but not all cases 



442 SERODIAGNOSIS. 

of paratyphoid fever will agglutinate equally well with para- 
typhoid bacilli isolated from different epidemics. There are 
now two well-recognized types of para-organisms. It is neces- 
sary, therefore, in case no agglutination is obtained with a 
typhoid bacillus, to secure members of both groups of the para- 
typhoid bacilli and to carry out agglutination tests with each. 
A high agglutination with any one of the varieties used indi- 
cates a probable infection with that variety of bacillus. The 
method of testing is similar to that described under Widal 
reaction. 

Infections Due to the Bacillus Cell Communis. — Genei al in- 
fections by the Bdcillus coli communis show a moderate agglu- 
tinating power in the blood for fresh twenty-four-hour cultures 
of the organism. 

Infections Due to Members of the Dysentery Group of 
Bacilli. — This group of bacilli contains at least three separate 
species, differing from each other in their agglutinations and 
reactions when grown upon sugar media. The bacillus origi- 
nally isolated by Shiga^ from cases of endemic dysentery in 
Japan has been shown to give a macroscopic agglutination with 
the blood of these patients in dilutions even as high as 1 to 
40 or 1 to 60 in an hour. 

The Flexner^ type of bacillus has also been found to agglu- 
tinate in low dilutions with the blood of patients from whose 
stools this bacillus has been isolated. 

Choleea. — A moderate number of tests have been carried 
out on the agglutinating reaction of serum from cholera patients 
on the specific bacillus, and such reactions have been found to 
appear at a fairly early period in the course of the disease, and 
develop in as high as 1 to 40 dilutions. 

A diagnosis can be made earlier and more certainly by the 
isolation of the bacilli from the stools and testing them with 
an immune serum of high agglutinating power. ^ 

Plague. — An agglutination of the plague bacillus has been 
observed beginning with the second week of the infection and 
rising later even as high as 1 to 40. The reaction, however, is of 



3 Deut. med. Wochen., 1901, p. 741. 

4 Bun. Johns Hopkins Hosp., 1900, vol. ii. 

5 Kolle and Gotschild : Zeit. f. Hygiene, 1903; Bd. xliv, p. 1.. 



AGGLUTINATION REACTION. 443 

little practical value, since the diagnosis can be made much 
earlier by the cultural isolation of the bacillus from the bubo or 
by the inoculation of susceptible animals.^ 

Malta Fever. — The serum of patients suffering from Malta 
fever usually gives a marked agglutination with the Micrococcus 
melitensis. The reaction has been seen to occur in a dilution as 
high as 1 to 50, or in some cases 1 to 300.'^ Using emulsions 
of dead bacteria in physiological saline solution, the agglutinat- 
ing power of the serum may rise to 1 to 1000 or more, if the 
mixture is left for twenty-four hours at room temperature.^ 

Mandelbaum's Test for Typhoid Fever.^ — Mandelbaum 
states that typhoid bacilli string out into long chains or the 
chains coil into a snarl when typhoid serum is added to a 
culture of the bacilli. This reaction seems to be specific, as 
he never observed it with any serum except that from typhoid 
patients. The reaction occurs in three or four hours and is 
readily perceptible in the hanging drop under the microscope 
by this time. This reaction may also occur long before the 
agglutination test gives positive findings. It also seems to occur 
with serum from persons with a history of typhoid in the past, 
although the reaction develops more slowly in such cases. The 
reaction was also slow but pronounced in a case of a chronic 
typhoid bacillus carrier. Mandelbaum has worked out a simple 
technic for the test, aiming to adapt it for general use. From 5 
to 8 cubic centimeters of a solution of 2 grams of sodium citrate 
in 100 cubic centimeters of ordinary bouillon is sterilized and a 
portion placed in a test-tube, and then this sodium-citrate-bouil- 
lon tube is inoculated with a loop of a motile typhoid-bacillus- 
bouillon culture or a scrap from an agar culture of typhoid 
bacilli. The culture must not be recent, but capable of growing 
well when further cultivated. A drop of blood is then obtained 
from the patient, aspirated into a long tapering capillary pipette 
with rubber cap like a medicine dropper. Then ten or fifteen 
times as much of the sodium-citrate-bouillon inoculated with the 
typhoid-bacillus cultures is drawn up likewise into the pipette. 
By releasing the pressure on the rubber cap, the fluid gradually 



G Martini : Zeit. f. Hygiene, 1902, Bd. xli, p. 159. 

7 Kretz : Wien. klin. Wochen., 1897, p. 1076. 

8 Basset-Smith : Brit. Med. Jour.. 1902, p. 861. 

• Miinch. med. Wochen., Jan. 25, 1910, Ivii. No. 4. 



444 SERODIAGNOSIS. 

rises to fill the lower third of the broader part of the pipette. 
The tapering tip is then fused and the pipette well shaken to 
mix the contents. The pipette is then kept at a temperature 
of 37° C. (98.6° F.) for from three to four hours. The sodium 
citrate addition is for the purpose of preventing coagulation, 
and after this interval the red corpuscles will be found collected 
on the bottom of the pipette, with a clear fluid above. The 
rubber cap is then taken off and a drop of the clear fluid is taken 
for examination as a hanging drop under the microscope. If the 
blood came from a person with typhoid the bacilli in the hanging 
drop will be adherent, while with non-typhoid serum the bacilli 
are scattered through the fluid. When the chains are found, 
and also a few isolated bacilli between the chains and snarls, 
the reaction is typical of typhoid existent several years 
before. In the one chronic typhoid-bacillus carrier he was able 
to examine, the reaction at the fourth hour was typical of a 
positive existing typhoid, but then the picture gradually became 
modified, so that by the end of the eighth hour the hanging 
drop resembled the findings in an old cured case. According 
to his experience, there are thus three forms of the reaction, 
which, however, is not very extensive — only 12 cases of existing 
typhoid, 16 of long-past typhoid, and 1 typhoid-bacillus carrier. 
The findings were constant in each class and were constantly 
negative in 75 other persons apparently free from a history of 
typhoid. The pipettes are those used in opsonic tests. 

Subsequent observations by W. Gaehtgens and W. Kamm^^ 
confirm the value of this test not only in the early stages of 
typhoid fever, but also in the detection of typhoid carriers. 

This is a matter of great importance, as heretofore it has 
been almost impossible to identify these dangerous persons by 
any simple means, as in them the Widal is often of no value, 
while a certain detection could only be by cultural studies of 
an extensive character, carried out with specimens of urine and 
feces. 

If the findings of these observers are further verified, this 
test of Mandelbaum will be a valuable addition to our labora- 
tory technic. 



10 Miinch med. Wochen., 1910, Ivii, Bd. 26. 



WASSERMANN AND NOGUCHI REACTION. , 445 

THE PRINCIPLES OF THE WASSERMANN AND NOGUCHI 
REACTIONS, AND THEIR COMPARATIVE VALUE TO 
THECLINICIAN.il 

Since the appearance of the original communications of 
Wassermann, Neisser and Bruck announcing a new method of 
syphilitic diagnosis, medical literature both in Europe and in 
America has been literally flooded with papers dealing v/ith this 
subject, and it is highly probable that no other single topic has 
received so much consideration from so many worl?:ers in the 
various fields of medicine. A very great deal of this work has 
been of the most careful and painstaking character. While the 
subject is a relatively narrow one the significance of a luetic 
infection is such that every aspect of the question of serum 
diagnosis has been subjected to the most careful scrutiny and has 
been investigated from both the clinical and experimental points 
of view, and now, after over three years, certain facts have been 
definitely determined. 

It is perhaps as well at the outset to say, however, that 
neither of the reactions to be described can be regarded as 
absolutely specific. 

The Wassermann reaction, based upon an ingenious prin- 
ciple, worked out by Bordet and Gengou, gives to the experienced 
laboratory worker a very satisfactory means of diagnosticating 
syphilitic affections, even in individuals who were infected years 
ago, of course excluding a dormant condition of patients at the 
end of a successful treatment. 

In order to be able to grasp the steps in the Wassermann 
reaction it is indispensable to be acquainted with the steps 
involved in demonstrating antibodies of any kind formed acci- 
dentally (by disease) or purposely (immunizing) in the body 
fluids of an animal. Ehrlich showed long ago that in the 
demonstration of antibodies, or, as he called them, amboceptors, 
three distinctly different substances are required in order to 
form a complete reaction : First, the cell or poison against which 
we wish to immunize, or, more plainly, against which we desire 
to obtain an antibody; second, the antibody (or amboceptor) 
obtained by repeated injections of the special cell or poison into 



11 Kaplan : Am. Jour, of Med. Sci., Jan., 1910. 



446 SERODIAGXOSIS. 

the rabbit (or any other suitable animal) ; third, a completing 
substance — the complement. This latter substance is present in 
variable quantities in the sera of all animals, its quantity being 
rather constant in guinea-pigs. It is destroyed by heating 
various sera to 56 or 57 degrees centigrade for one-half hour, and 
is similarly affected by various other physical agents. This is 
not the case with antibodies which are comparatively thermo- 
stable. AYe have then three factors: (1) A cell to be destroyed 
or a poison to be neutralized; (2) a substance capable of doing 
this — the amboceptor or antibody; and (3) a completing sub- 
stance, without which the reaction cannot take place — ^the 
complement. 

Ehrlich and others in order to impress the reaction upon 
the minds of men interested in immune processes, made use of 
diagrams. To make it still more familiar, let a lock represent 
the cell, a key which fits it the antibody, and the hand that will 
turn the key the complement. By giving a lock to a smith we 
can get a key made to fit the specific lock exactly. When we 
inject cells we can get an antibody which exactly fits the cell 
injected, and the same is true when we inject a bacillus or a 
poison. All these substances capable of producing antibodies 
(antibody generators) are known as antigens. 

To determine whether a bacterium was killed or a poison 
neutralized by being exposed to the action of a specific ambo- 
ceptor, is not as simple a process as the demonstration of the 
destruction of red blood-corpuscles by an amboceptor directed 
against them. A suspension of red blood-corpuscles minus 
amboceptor and complement gives an opaque red mixture; when 
we add the amboceptor plus complement and incubate at 37° 
C. the opacity disappears and a clear red fluid results. It 
is apparent that hemolysis or destruction of red cells is a 
phenomenon that can readily be seen in vitro, and its presence 
signifies that the three substances spoken of above are present in 
the test-tube. If any one of the three is not there, or is present 
in an inactive state, the red cells will remain unaffected and the 
mixture will retain an opaque, red color. 

Hemolysis. — The phenomenon known as hemolysis depends 
upon the destruction of red blood-corpuscles. There are many 
reagents capable of doing this, such as distilled water, acids, and 



WASSERMANN REACTION. 447 

alkalies. It is also possible to form in warm-blooded animals 
substances which will bring about hemolysis against certain red 
blood-cells. This is accomplished by injecting an animal (a 
rabbit or goat) with the cells of a sheep or any other animal. 
The serum from such a rabbit, when brought in contact with the 
cells of a sheep will cause the mixture to become clear (hemo- 
lysis). The same amount of serum from an untreated rabbit 
will have no effect on a similar suspension of sheep cells. The 
substance produced in the rabbit's serum is known as the anti- 
sheep amboceptor, and together with sheep cells and complement 
(from a guinea-pig) is known as a hemolytic system. 

We learn from the above exposition that in order to prove 
the presence or absence of certain antibodies, we make use of 
the phenomenon of hound or unhound complement, utilizing a 
hemolytic system simply as an indicator. Exactly the same prin- 
ciple is applied to the serum diagnosis of syphilis. 

Luetic Antigen. — Unable to produce a growth of spirochsete 
pallida upon any culture medium, we have to be contented with 
organic extracts containing them in greatest numbers, for this 
purpose the liver of the luetic fetus fills the requirement. The 
extract obtained is known as leutic antigen, and need not con- 
fuse an3^body, for we know that an antigen is a body capable of 
forming antibodies. If an individual has had syphilis some 
years ago, he would also have syphilitic antibodies in his serum 
which, when brought in contact with the extract from the syphi- 
litic liver, would invariably bind complement, and a hemolytic 
system (sheep cells or any other red cells with the corresponding 
amboceptors) will not be affected, because the complement had 
been bound previously. 

Principle and Technic of the Wassermann Eeaction. — As 
mentioned before, antibodies will attract complement if the 
antigen responsible for their formation is present in the same 
test-tube. In the Wassermann reaction a serum containing anti- 
bodies capable of uniting with the antigen is used (substance 
containing lipoids) and thus deviating the introduced comple- 
ment, will not permit hemolysis to occur, if sheep cells and their 
antisheep amboceptor are subsequentlv placed in the same tube, 
as for obvious reasons, the complement was bound or deviated 
previously by conditions suitable for such an interaction. If 



448 SERODIAGXOSIS. 

the patient's serum does not contain the required antibody, the 
introduced complement will remain unbound and in a fit condi- 
tion to destroy the sheep cells when subsequently introduced 
with their antisheep amboceptor. 

Modus Opeeaxdi. — (1) Obtaining blood from patient: A 
fairly stout piece of rubber tubing is placed a little above the 
elbow and held snugly in place by an artery clamp. Do not 
obliterate the pulse. This brings into prominence the veins at 
the bend of the elbow. To a stout hypodermic needle (use a 
19 bore — -one and one-half inch needle) attach a two-inch piece 
of rubber tubing. Holding the free end of the rubber tubing 
in an ordinary sterile test-tube, quickly plunge the needle into 
the most prominent vein; if expertly done, the patient will 
hardly feel it and the blood will immediately begin to flow. 
About 6 to 10 cubic centimeters of blood is withdrawn and 
placed in the ice box over night to coagulate. The serum 
separates and may be pipetted off absolutely clear without cells. 
It is advisable to take the blood as far from a meal as possible, 
as proximity to a meal makes the blood lipemic, interfering with 
perfect working conditions. (2) Having obtained 1 or 2 cubic 
centimeters of clear serum, it is placed in a test-tube in the 
thermostat at 56 degrees for one hour*. Care must be taken not 
to permit the heat to rise too high (over 58 degrees). (3) 
After this, 0.2 cubic centimeter is placed in each of two test- 
tubes, one the test, the other the control. (4) To each is now 
added 0.1 cubic centimeter fresh complement. (5) To the test 
portion is added one unit of antigen. The control does not 
receive any antigen. (6) Each tube receives now 3 cubic centi- 
meters of a 0.85 per cent. NaCl solution. In order to be able 
to judge properly the correctness of the procedure, the more 
controls one has the better ; it is therefore necessary to compare 
the serum to be tested with two sera from known positive and 
negative bloods. (7) Shak:e every tube well and place in incu- 
bator at 37 or 38 degrees for one hour. During this time, if the 
serum is luetic the antibodies present will, together with the 
antigen, bind the complement and render it inactive for hemoly- 
sis. (8) After one houi^ incubation each tube receives two units 
of amboceptor and 1 cubic centimeter of a 5 per cent, suspension 
of sheep cells in 0.85 ^NTaCl. The tubes are again vigorously 



NOGUCHI REACTION. 449 

shaken and placed in the incubator at 37 degrees and inspected 
after ten minutes. If the reagents are properly adjusted hemoly- 
sis begins in the control tubes in fifteen to twenty minutes^ and 
careful watching becomes a very essential point at this stage of 
the test. As soon as the control is completely hemolyzed the 
tubes are to be compared; only those should be pronounced 
negative that show a transparent fluid the same as the control. 

Permitting the tubes to stand undisturbed in a cool place 
(15 to 17 degrees C.) for tw^enty-four hours shows in the positive 
test a deposit of red cells, the size of the deposit depending upon 
the severity of the infection or proximity to the initial lesion as 
well as upon the degree of balance of the reagents used. Usually 
a markedly positive serum gives at the end of twenty-four hours 
a clear supernatant fluid of a light pink hue with a Bordeaux red 
accumulation on the bottom of the tube. The weaker the 
reaction, the redder the supernatant fluid and the scantier the 
deposit of cells. In testing more than one serum, the reaction in 
each individual test must be considered as finished as soon as the 
controls are completely hemolyzed, in which case the two tubes 
are immediately removed to a cool place. 

Principle and Technic of the Noguchi Reaction. — This is 
same as in the Wassermann, excepting that the amboceptor is 
directed against human cells. It also facilitates the handling of 
reagents, as they are mostly paper soaked in the antigen and 
amboceptor. These do not readily deteriorate, as is the case 
with fluid biological reagents. The serum does not need inacti- 
vation at 56° C. 

Modus Operandi. — (1) With a capillary pipette allow one 
drop of fresh serum to fall into a narrow (1 centimeter 
lumen) test-tube. The pipette is not to be used for any other 
serum. (2) Add 0.05 cubic centimeter fresh complement. (3) 
To the front row (rear row for control) add one piece (more or 
less, depending on the titre) of antigen paper. (4) Prepare a 
suspension of human cells 1 drop of blood to -1 cubic centimeters 
NaCl 0.95 per cent. It is best to prepare about 60 cubic centi- 
meters of ¥aCl solution and allow 15 drops of blood to fall from 
the experimenter's finger into the solution. The huuian cell 
suspension is placed over night in the ice box. N'ext morning 
the supernatant clear salt solution is pipetted off and a fresh 

29 



450 SERODIAGNOSTS. 

quantity of N'aCl is added (about 55 cubic centimeters) to the 
cells in the beaker. Of this cell suspension add 1 cubic centi- 
meter to every tube in the rack. (5) Incubate for three-quarters 
or one hour at 38 or 39 degrees, preferably in a large dish of 
warm water. Occasionally shake the tubes, to insure proper 
solution of the biological substance on the antigen paper. (6) 
Add to each tube (after incubation), front and back rows, one 
piece of amboceptor paper more or less, the quantity depending 
on the titre) and replace in the incubator, observing the result 
after ten minutes, and watching carefully the controls. 

- It will be noted in about fifteen minutes, more or less, that 
the rear row begins to get clear, and when complete transparency 
is obtained the test and control tube are to be removed to a cool 
place and observed. If the reaction is positive, then the front 
tube (test) will be opaque, in marked contrast to the control, 
which is transparent. For convenience of observation, make 
use of a fine sealed tube (about 1 millimeter in diameter) filled 
with black ink, which, when placed behind the control, will 
appear as a clear black line, whereas the positive tube will not 
show the black line, or it appears as a dim shadow — depending 
on the strength of the reaction. 

It has been stated that a positive Noguchi test and a 
negative Wassermann is often due to the presence in the patient^s 
serum of antisheep amboceptors. It is not necessary to perform 
this test with every serum as a control. Only sera giving the 
above results need be subjected to a verification. To demonstrate 
the antisheep amboceptor, place 1 cubic centimeter of a 5 per 
cent, suspension cff sheep cells in a test-tube, add 0.2 cubic centi- 
meter of patient^s serum and 0.1 cubic centimeter complement, 
add 3 cubic centimeters of NaCl solution, place in incubator, 
and observe. If the amboceptor is present, the cells will dissolve 
and the mixture become transparent. The time consumed 
depends upon the number of amboceptor units present. 

CoNTEOLS. — In the Wassermann and N'oguchi reactions it 
is of vital importance to have every possible error exciaded. 
The substances to be controlled are the antigen, the amboceptor, 
and each individual serum. 

Tlie Antigen Control. — This biological reagent, as is known, 
can per se inhibit hemolysis. To measure the degree of such 



NOGUCHI REACTION. 451 

interference, a tube containing a well-known normal serum, plus 
antigen, plus complement, and antisheep amboceptor plus sheep 
cells ought to hemolyze in about twenty to thirty minutes. No 
reaction is to be considered as finished before the antigen control 
tube is completely hemolyzed. 

Tlie Amhoceptor Control. — Upon the efficiency of the anti- 
sheep amboceptor depends the rapidity of hemolysis of the sheep 
cells. It is therefore necessary to establish the amboceptor effi- 
ciency in a separate tube containing sheep cells, plus complement, 
plus antisheep amboceptor. It is not essential to add normal 
serum. The tube containing the above ingredients is always the 
first to hemolyze, requiring about fifteen to twenty minutes for 
a complete hemolysis. 

Control for Each Serum. — Every serum more or less has 
the power to interfere with hemolysis to a slight degree. In 
order to control the factor of individual inhibition, every serum 
tested is placed in each of two tubes, the front tube contains the 
antigen and all other biological reagents, the rear tube receives 
everything but the antigen. This shows the degree of individual 
inhibition as compared with the tube containing the amboceptor 
control. 

Efficiency of the Entire System. — For this a well-known 
luetic serum is utilized. The reaction is to be positive, and 
hemolysis should not occur in the front tube, even if exposed to 
incubation temperature for hours after the controls hemolyzed. 

Equipment. — At least one dozen or even more of Mohr's 
pipettes, 1 cubic centimeter, graduated into /4oo- One dozen 10 
cubic centimeter pipettes, graduated into %o- One gross of 
ordinary test-tubes. One gross of test-tubes 1 centimeter in 
diameter, 12 centimeters high. One-quarter dozen of graduated 
cylinders, 50 cubic centimeters; one-quarter dozen 100 cubic 
centimeters. Two 50 cubic centimeters measuring flasks with 
glass stoppers. A few pounds of glass tubing, 5 millimeters 
bore, to make capillary pipettes. One-half dozen test-tube racks 
for Wassermann tubes; one-half dozen test-tube racks for 
Noguchi tubes. A piece of rubber tubing for tourniquet. One 
artery clamp for above. One dozen hypodermic needles, 19 bore. 
One thermostat regulated at 57 degrees and one reonlated at 37 
degrees. One electric centrifuge. Labels and pencil for writing 



452 SERODIAGNOSIS. 

on glass. One tall glass jar for flushing through used pipettes, 
height to be greater than any pipette used. One dozen Petri 
dishes. One dozen beakers, 100 cubic centimeters capacity. Two 
fine forceps, and two Hagedorn needles. One package of quanti- 
tative filter paper. One razor (for killing guinea-pigs). One 
15 cubic centimeter Luer syringe. 

Preparation of Animals. — Antisheep Amboceptors: 
Several healthy rabbits (not less than four) receive every fifth 
day 1, 2, 3, 4 and 6 cubic centimeters of well-washed sheep cells. 
This number of rabbits is used, as one or two may die during the 
injection weeks. The cells are obtained from the slaughter 
house and immediately defibrinated with a wire defibrinator or 
glass beads. In order thoroughly to wash the cells a high speed 
centrifuge is necessary, capable of making at least 3000 revolu- 
tions to the minute. Two of the centrifuge tubes are filled with 
the fluid sheep blood, it being advisable, in order not to spoil the 
centrifuge, to have them of equal weight. The first centrifugali- 
zation brings the cells to the bottom, and the clear supernatant 
serum is pipetted off. The cells are now mixed with 0.95 per 
cent. NaCl solution and centrifugalized again, and the super- 
natant clear fluid is again pipetted off; this is repeated three 
times. The cells are now approximately serum-free. The entire 
quantity of cells in the tube is now brought up to its original 
volume with 0.95 per cent. NaCl solution, and of this, 2 cubic 
centimeters is used for the first injection. With a sterile glass 
syringe this quantity is injected into the peritoneal cavity, having 
previously shaved and cleaned the puncture area. Cotton and 
collodion prevent wound infection. This procedure is repeated 
five days later with 4 cubic centimeters of cells brought to its 
original volume, etc., until each animal has been injected five 
times. Mne days having elapsed since the fifth injection, the 
serum of the rabbit contains now a high lytic power against the 
red blood-corpuscles of the sheep. The rabbit is killed and its 
serum used. 

In the Wassermann reaction 1 cubic centimeter of a 5 per 
cent, suspension of well-washed sheep red cells in 0.95 per cent. 

NaCl solution is the standard dose for each test. It is evident, 
therefore, that in order to test the power of our rabbit serum 

(antisheep amboceptor, as it is- now called) we must use this 



PREPARATION OF ANIMALS. 



453 



quantity of sheep cells. Into each of six test-tubes is placed 1 
cubic centimeter of a 5 per cent, suspension of sheep cells in 
XaCl 0.95 per cent. These are marked from 1 to 6, and to each 
is added 0.1 cubic centimeter of fresh guinea-pig serum (this is 
Imown as the complement serum and is the quantity used in the 
Wassermann test). We now add to test-tube (1) 1 cubic centi- 
meter of a 1 to 200 solution of our amboceptor. To test-tube 
(2) we add 1 cubic centimeter of 1 to 400; to test-tube (3), 1 
cubic centimeter of 1 to 800; to test-tube (-4), 1 cubic centimeter 
of 1 to 1600; to test-tube (5), 1 cubic centimeter of 1 to 3200. 
This is placed into the thermostat at 37 degrees and the result 
noticed after fifteen minutes, thirty minutes, up to two hours. 
It will be seen that in fifteen minutes test-tube (1) is clearing 
up, or is clear (hemolysis) ; this would indicate that 1 cubic 
centimeter of a 1 to 200 solution of our amboceptor is capable 
of destroying in fifteen minutes 1 cubic centimeter of a 5 per 
cent, suspension of sheep cells. This proportion — 1 to 200 — is 
too strong, and may give negative results with some positive sera. 
The unit strength of the antisheep amboceptor is usually twice 
the quantity capable of hemolyzing the 1 cubic centimeter of 
cells in two hours. If 1 to 1600 shows hemolysis after two hours 
and 1 to 3200 does not, then 1600 divided by 2 is the strength 
of the amboceptor, one unit equals 1 to 800. It is best to run 
two series of titration — one like the above, the second beginning 
with 1 to 250, 1 to 500, 1 to 1000, etc., so that a proper mean 
can be established and a more exact unit made. In table form 
the above is expressed as follows : — 



Tube 1 amboceptor = 1 to 200 
"2 " = 1 to 400 

" 3 " =1 to 800 

" 4 " =1 to 1600 

" 5 " =1 to 3200 



Complement 0.1 
0.1 
0.1 
0.1 
0.1 



Sheep cells. 



5"^ 1 c.c. 



Cells hemolyzed in 



15 minutes 
30 minutes 
50 minutes 
1^2 hours 
2 hours 



Strengfth of 1 unit, 1 to 1600 ; dose for 1 test. 1 to 800. Date. 



A full-grown rabbit usually furnishes from 50 to 60 cubic 
centimeters of serum. This is to be kept in a glass-stoppered 
flask in the ice box (lower compartment). The hemolytic power 
does not indefinitely remain the same as in the beginning; it is. 



454 SERODIAGNOSIS. 

therefore, necessary to establish the titre at least once every 
week, and to make up the dilutions accordingly. These dilu- 
tions are to be prepared on the day of testing. The rabbit serum 
does not have to be inactivated to get rid of the complement in 
it, as the quantity of serum used is too small to influence in any 
way the resulting outcome of the test. 

Preparation of Complement. — A full-grown guinea-pig is 
held over a Petri dish, and after having it narcotized, the blood- 
vessels of the neck are severed with a razor. Suspended by the 
hind legs the animal is exsanguinated, and the collected blood is 
permitted to remain at room temperature for at least three 
hours. The serum collects in large drops and may be pipetted 
off, or the coagulum plus the serum is placed in a centrifuge 
tube and after five minutes centrifugalization the supernatant 
serum is pipetted off into a sterile test-tube; but such a serum 
is not as reliable as when left for three hours with its cells. 
About 6 cubic centimeters of complement is obtained from one 
guinea-pig. 

Preparation of Sheep Cells. — Obtained from the slaughter 
house, the cells are washed three times with 0.95 per cent. N'aCl 
solution, and 1 or 2 cubic centimeters is mixed with 20 or 40 
cubic centimeters of salt solution, making a 5 per cent, suspen- 
sion of cells. 

Preparation of Antigen. — The fresh liver of a luetic fetus 
or the liver of any baby cadaver is chopped up very finely, and 
the mass is spread on a few Petri dishes and dried. The drying 
process is hastened by a current of air produced by an ordinary 
electric fan. Lately, not only baby livers, but also the livers of 
dogs, the hearts of guinea-pigs, and other organs were used to 
make antigen. The usefulness of the antigen is only established 
when in actual standardization it is found serviceable and works 
faultlessly with decidedly syphilitic and unquestionably normal 
sera. Consequently it makes little difference whether one uses 
the extract obtained from the liver of a syphilitic fetus or from 
the heart of a guinea-pig, provided they are well titrated. 

It is better — according to German workers — to use more 
than one extract, and have a series with well standardized luetic 
liver antigen, one with guinea-pig heart, and another with dog 
liver, or normal human liver. To proceed with the making of 



PREPARATION OF ANIMALS. 



455 



antigen the obtained dried liver is rubbed into a powder and kept 
in an exsiccator over CaCl2 in a cool, dark place. According to 
Tscliernogubow, such, a powder is serviceable for a very long 
time. Of this powder, 0.5 gram is extracted at room tempera- 
ture or in an ice box with 25 cubic centimeters of 95 per cent, 
alcohol for twenty hours, then filtered, and the filtrate used for 
experiments. 

For the actual Wassermann test, one part of this opalescent 
filtrate is diluted with five parts of 0.95 per cent. NaCl, and 0.5 
to 1 cubic centimeter used for each test-tube, the dose depending 
upon the established titre. The above process extracts from the 
liver substances soluble in alcohol, chiefly bodies of a fatty nature 
(lipoids). There are other means of obtaining lipoids, the above 
being one of the simplest, having also in view the preservation 
of the antigen in an active form. The liver, instead of being 
dried and powdered, may be directly extracted with five volumes 
of absolute alcohol, and the extract obtained by driving the 
alcohol off at a temperature not higher than 40° C. or with the 
electric fan. The obtained extract is much more powerful than 
the above, is soluble in ether, from which NaCl solutions are 
made for use. The titre is established carefully as follows : 

Titration of Antigen. — The unit dose of antigen must be of 
such a strength that one unit will completely inhibit hemolysis 
of 1 cubic centimeter of a 5 per cent, suspension of sheep cells, 
with 0.2 cubic centimeters of a known luetic serum plus 0.1 cubic 
centimeter of complement; provided double this dose does not 
interfere with the complete hemolysis of cells using a known 
normal serum and complement. 



Table of Antigen Standardization. 





Luetic Series 


Normal 


Series 


Each 


tube contains syphilitic serum, 


Each tube contains 


normal serum. 


0.2; complement, 0.1; 


cells, 1 c,c. 5fo; am- 


0.2, complement, 0.1; 


cell 


s, 1 c.c, 5'i; am- 


bocepter, 


2 units. 




boceptor, 2 units. 
















Hemolysis 


Tube 1, Antigen, 0.025 


1 hour 15 min. 


Antia-en, 0.025 




15 minutes 


" 2. 


0.05 


1 " 25 " 


" 0.05 




15 " 


" 3, 


0.075 


2 " 50 " 


0.75 




15 


" 4, 


0.10) 


No hemolysis af- 


" 0.10 




20 


" 5. 


0.15 > 


ter 24 hours in 


" 0.15 




30 '* 


" 6, 


0.23) 


incubator. 


*• 0.20 




35 



Dose of 1 unit 0.1 cc. 



456 SERODIAGXOSIS. 

From the above facts it is evident tliat the dose next to the 
largest heniol3'zing dose is tlie strengtli of one nnit^ or 0.1 cubic 
centimeter. It is also apparent that 0.2 cubic centimeter, or a 
double dose, will not inhibit hemolysis when used with a normal 
serum. 

In establishing the unit dose of antigen as well as antisheep 
amboceptor it is of utmost importance to titrate two or three 
times in order to get as uniform results as possible, and only 
uniform work will enable one to come to a proper conclusion as 
to which is the necessary dose. For establishing the strength of 
the antigen and amboceptor, well-known fresh luetic and normal 
sera are to be used, as well as fresh suspension of cells and fresh 
complement. 

Before using the standardized reagents it is advisable to 
perform two or three actual tests with well-known positive and 
negative sera. After this the substances may be considered safe 
for use. The above lines will give one a fair idea concerning the 
preparation of the biological reagents for the Wassermann 
reaction. For the Xoguchi reaction it will be necessary, first, 
to acquaint the reader with the principles involved, and then the 
preparation of reagents will follow. 

Preparation of Eeagexts. — Antiliuman Amboceptor: 
Eabbits are injected with human cells the same as the sheep 
rabbits. After nine days the rabbits are killed, their serum 
collected and disposed of as follows : The fluid amboceptor loses 
strength on standing, so much so that it may not contain one- 
fourth of its original power a month after the first titration. 
In a dry state it can be used for a very long time without 
losing its strength. Prepare antiliuman amboceptor by cutting 
quantitative filter paper in 5 millimeter squares. These squares 
are stuck onto pins fastened to a cardboard. With a very fine 
capillary pipette (as fine a one as can be made) one drop is blown 
on each piece of filter paper and placed in the thermostat for 
dr3'ing. In half an hour the papers are dry and fit for use. By 
this method each square receives exactly the same quantity of 
serum, and is not subject to differences in dissemination which 
must be considered when the serum is blown on a larger piece 
of filter paper and cut subsequently in 5 millimeter squares. The 
method takes longer, but the difference is worth while, for each 



THE NOGUCHI TEST. 



457 



square holds exactly the same quantity of amboceptor. In the 
test one piece of this amboceptor is serviceable. 



Titration op Antihdman Amboceptors. 



Tube 1, Amboceptor, 3^ piece 
" 2, " 1 

" 3, " IK •' 



Human cells 1 drop 
to 4 c.c. NaCl. 



1 c.c. 

1 c.c. 

1 c.c. 

1 c.c. 



Complement. 



0.05 
0.05 
0.05 
0.05 



Hemolysis in 



2 hours. 
20 " 
11 " 



Value of the Noguchi Test. — Workers with the Wasser- 
mann reaction often could not explain why a positive result 
could not be obtained with some true luetic sera. Later it was 
demonstrated that this was due to the presence in the human 
serum of substances capable of dissolving the red blood-corpuscles 
of the sheep; in other words, some human sera contained anti- 
sheep amboceptors. 

Sometimes the quantity of antibody is so small that a goodly 
portion of the complement escapes unbound and does its work 
by bringing about partial hemolysis; that is the condition of 
affairs taking place in some weak reactions. If such a serum 
contained antisheep amboceptors, they would have enough com- 
plement to cause hemolysis and render the result negative. 

In the Noguchi test this cannot take place, for the hemoh^tic 
system used consists of human cells plus antihuman amboceptor, 
and, naturally, the human organism does not contain antihuman 
amboceptors. Luetic sera containing antisheep amboceptors will 
give a negative Wassermann but a positive Noguchi test. 

As a result of much work with the Wassermann and 
Noguchi tests, Kaplan is able to give a fair opinion as to its 
uses. Shortly stated, the two reactions are of the foremost 
importance to the clinician, and so far as accuracy is concerned, 
they almost occupy the first place among our means of detecting 
diseases. 

The Wassermann reaction gives a negative result in 8 or 9 
per cent, of syphilitic sera. This rather undesirably high per- 
centage of error is reduced to 1 to 1.5 per cent, when using the 
Noguchi and the Wassermann combined. 



458 SERODIAGNOSIS. 

Never render a decision after one test. Always perform two 
Wassermann and two ISTognchi tests on different days, using the 
same serum. It is also to be borne in mind that a fairly 
marked Wassermann reaction, 99 times out of 100, means 
syphilis, and that a negative Noguchi the same number of times 
means no syphilis. The two methods are very decisive, but in 
opposite ways; and used together, carry with them an assurance 
which no amount of thoroughness and precision will replace if 
only one method is used. 

The laboratory worker, being responsible to the clinician 
for his statements, ought to be in a position to help him con- 
siderably, but only when his work has been carried out very 
carefully, unbiased by personal opinion, and submitting the 
result of a delicate test as read from the test-tube. 

It is advisable to work with both methods, the Wassermann 
and Noguchi, as one is a check on the other, which, if properly 
performed, should give correct reports in 98 per cent, of cases. 

Questionable reactions are not to be used for diagnosis, and 
if a serum does not react strongly after a number of repetitions 
of the test, the diagnosis is to be left to the clinician. 

Exceptionally strong reactions are obtained in untreated 
cases of general paresis with both tests, as well as in primar}^ 
sores four weeks after infection. 

The Hecht-Weikberg-GtRAdwhol Test. — Massed statis- 
tics of Wassermann workers have clearly shown that, following 
the technic of Wassermann, employing the most exact methods 
of titrating all reagents, and using the most sensitive antigens, 
the results are not uniformly positive in all cases of evident 
clinical syphilis. Other tests have been submitted in an effort 
to check and correct if possible this uncontrollable and often 
unexplained error. It has been noted that sheep amboceptor 
(common in most humans) existing coincidently with a low 
amount of syphilitic antibodies is a combination which accounts 
for some negative Wassermanns despite the actual presence of 
syphilis. 

The Hecht- Weinberg test uses the natural emboceptor in 
the unheated human sera and is said to give 10 per cent, more 
positive reactions in presence of syphilis than the straight 
Wassermann technic. 



TESTS. 459 

TecJinicA^ — Place in a rack 14 small test-tubes. The first 
ten of these are used to determine the hemolytic index of the 
suspected blood. The last four tubes are used for the actual 
test. Add 0.1 cubic centimeter of fresh unheated patient's 
blood-serum to each of the first ten tubes, then add decreasing 
amounts of normal salt solution to each of the 10 tubes, begm- 
ning with 1.0 cubic centimeter, then 0.9; 0.8; 0.7; 0.6; 0.5; 0.4; 
0.3; 0.2; 0.1 — to the succeeding 9 tubes. Next add increasing 
amounts of fresh, 5 per cent, suspension of sheep's cells starting 
with 0.1 cubic centimeter and ending with 1.0 cubic centimeter. 
Place rack in water bath for one-half hour. The tube which 
last shows complete hemolysis constitutes our "hemolytic index." 

If this tube is tube 4, then the index is 4, because this tube 
had received 0.4 cubic centimeters of sheep's corpuscles. The 
index determines the amount of sheep's corpuscles to be added 
to the remaining tubes. 

The first three of these (Nos. 11, 12 and 13) constitute the 
tubes of the actual test, while the last tube in the rack (No. 14), 
serves as the serum-control tube. Tubes 11, 12 and 13 receive 
therefore the patient's serum, the proper amount of sheep's cor- 
puscles (dependent upon the hemolytic index) rising strengths 
of antigen, but no complement and no amboceptor. Tube 14 
receives only sheep's corpuscles' but no antigen. The usual 
technic is to use 0.1 cubic centimeter of a diluted antigen deter- 
mined by titration in tube No. 11, 0.15 cubic centimeter in tube 
No. 12 and 0.2 cubic centimeter in tube No. 13. In order to 
equalize the volume of fluid in these 3 tubes add 0.2 cubic 
centimeters of normal saline to tube No. 11, 0.15 cubic centim- 
eter to tube No. 12 and 0.1 cubic centimeter to tube No. 13, 
and to tube No. 14, 0.3 cubic centimeter. The tubes are then 
agitated and placed in a water bath for one-half hour. These 
last 4 tubes are filled at the time the additions are made to the 
first 10 tubes, and left with them in the water bath for one-half 
hour for fixation to complement. The rack is then talvcn out 
and the hemolytic index computed. If the index is low, say 
from 1 to 4, add 0.1 cubic centimeter of sheep's blood to the last 
4 tubes. If the index is from 5 to 7 use 0.15 cubic centimeter 



12 R. B. H. Gradwohl : Jour. A. M. A., Ixiii, No. 3, 241, 1914. 



460 SERODIAGNOSIS. 

and if between 7 and 10 use 0.2 cubic centimeter of sheep's 
blood. 

GradwohFsi^ i^ experience shows this index never to be 
over 10. If the patient's serum has an index below three it is 
doubtful value; if above three it may be regarded as absolute. 
The reaction is read off exactly as is the Wassermann, that is 
inhibition or non-hibition of hemolysis. According to Grad- 
wohl, the Hecht-Weinberg test has never been found negative 
in any blood-serum which showed a positive Wassermann, but 
has frequently been found positive in blood serums showing a 
negative Wassermann. 

Luetin Heaction. — Noguchi first announced his cutaneous 
reaction for syphilis in 1911.^^ Its chief value lies in the fact 
that it aids in the diagnosis of this condition as soon as the 
infection has gained entrance into the system and often ante- 
dates the occurrence of the Wassermann reaction. His material 
is prepared as follows : Pure cultures of the Treponema pallidum 
(see page 175) are allowed to grow for periods of six, twelve, 
twenty-four, and fifty days at 37° C. under anaerobic conditions. 
They may be cultivated in ascitic fluid containing a piece of 
sterile placenta, or in ascitic fluid agar also containing 
placenta. The lower portion of each solid culture is cut out 
and the tissue removed. These agar columns, containing large 
numbers of spirochetes, are carefully ground in a sterile motor. 
This paste is then gradually diluted by adding, little by little, 
the fluid culture, until the emulsion becomes perfectly liquid. 
This mixture is heated to 60° C. for one hour in a water-bath 
and 0.5 per cent, carbolic acid is added. When this mixture is 
examined under the dark-field microscope 40 to 100 pallidse may 
be seen in each field. This suspension is called Luetin. 

Principle of the Test. — The skin of animals repeatedly 
inoculated with Spirocliceta pallida and the skin of human beings 
suffering from syphilis do not behave in the same way as does 
the skin of non-syphilitic individuals. The skin of the syphilitic 
subjects reacts with an inflammation to the inoculation of the 
pallida substances (the luetin) prepared from the killed pure 



13 R. B. H. Gradwohl: Jour. A. M. A., Ixiii, 7, p. 514, 1914. 

14 M. L. Heidingsfeld : N. Y. Med. Jour., April 8, 1916. 

15 Jour, of Exp. Med., vol. xiv, 1911, p. 557. 



TESTS. 461 

cultures, while the non-syphilitic skin does not. The intensity 
of the inflammatory reaction produced by the luetin may vary 
from an inflammatory nodule to a pustule formation, lasting, 
as a rule, for several days. In some instances the reaction may 
commence as late as three or four weeks after the inoculation. 

Technic of Application. — The skin of the upper arm is 
sterilized with alcoholic sublimate solution before the injection. 
The amount of luetin injected is 0.05 cubic centimeter. This 
injection is intradermic, that is, in the skin, as superficially as 
possible. In non-infected cases there appears, after twenty-four 
hours, a very small, erythematous area at and around the point 
of injection. No pain or itching is experienced. This slight re- 
action gradually recedes within forty-eight hours and leaves no 
induration. In certain cases the reaction may reach a stage of 
small papule formation after twenty-four or forty-eight hours, 
after which time it commences to recede. 

The Eeaction. — In positive cases the following types of 
reaction occur: — 

(a) Papular Form. — A large, raised, reddish, indurated 
papule, usually 5 to 10 millimeters in diameter, makes its ap- 
pearance in twenty-four to forty-eight hours. The papule may 
be surrounded by a diffuse zone of redness and show marked 
telangiectasis. The dimensions and the degrees of induration 
slowly increase during the following three or four days, after 
which the inflammatory processes begin to recede. The color 
of the papule gradually becomes dark bluish red. 

(&) Pustular Form. — The beginning and course of this 
reaction resemble the papular form until about the fourth or 
fifth day, when the inflammatory processes commence to pro- 
gress. The surface of the indurated round papule becomes 
mildly edematous, and multiple miliary vesicles occasionally 
form. At the same time a beginning central softening of the 
papule obtains. Within the next twenty-four hours, the papule 
changes into a vesicle, filled at first with a semiopaque serum, 
that later becomes definitely purulent. Soon the pustule 
ruptures. 

(c) Torpid Form. — In rare instances the injection sites 
fade away to almost invisible points within three or four days, 
so that they may be passed over as negative reactions. Some- 



462 SERODIAGNOSIS. 

times these spots suddenly light up again after ten days or so 
and progress to small pustular formation. 

No marked constitutional s}Tnptoms have been observed 
after the use of the luetin. In most positive cases a slight rise 
in temperature takes place^ lasting for one day. 

Specificity. — Speaking summarily (iSToguchi), the follow- 
ing facts have been established : — 

1. The luetin reaction is specific for syphilis. 

2. The reaction is present in the majority of cases of ter- 
tiary, latent, and hereditary syphilis. 

3. It is less constantly present in secondary untreated and 
primary cases. 

4. In treated secondary cases the reaction is present in 
most instances. 

5. In general paralysis and tabes dorsalis the reaction is 
inconstant, but a positive reaction was obtained by Moore and 
Noguchi in about 60 per cent, of cases. 

6. In certain cases of tertiary and hereditary syphilis there 
may be a considerable inflammatory reaction at the site of 
injection of the control fluid, and the reaction may sometmies 
be as strong as that produced at the luetin inoculation site. 

7. The condition of the skin which gives the luetin reaction 
remains but little influenced by the antisyphilitic treatment, 
although a positive reaction can no longer be obtained in some 
cases which had been thoroughly treated and believed to be cured. 

The relation between the Wassermann and the luetin re- 
actions may be defined here in order that the fullest possible 
benefit may be derived from the use of both reactions. The 
Wassermann reaction is more constantly present than the luetin 
reaction in cases of primary and secondary syphilis, especially 
when only a slight amount of treatment or none has been given. 
On the other hand, the luetin reaction is more constantly pres- 
ent than the "Wassermann reaction in cases of tertiary and latent 
syphilis. Besides, in cases in which the Wassermann reaction 
and clinical manifestations of syphilis are very marked and the 
luetin reaction negative, an energetic treatment can reverse the 
situation completely. 

Thus, through the treatment the Wassermann and clinical 
symptoms gradually are made to disappear, while the luetin re- 



MEIOSTAGMIN REACTION. 463 

action becomes more distinct and the condition that gives the 
reaction to the Inetin persists afterward, probably until a cure is 
effected. From the above-cited facts it may be concluded that 
the luetin reaction possesses greater diagnostic value than the 
Wassermann in tertiary and latent syphilis, and also a decided 
prognostic value which the Wassermann does not. 

Caution. — While the application of the luetin reaction is 
simple enough to be within the reach of any physician, one must 
not underestimate the technical precautions necessary for obtain- 
ing reliable results, because all kinds of irregularity of the 
reaction can be obtained by a neglectful and faulty technic. 
Besides, it is important to become quite familiar with the reac- 
tions before one can recognize the milder form of positive 
reaction and avoid a misinterpretation. 

Meiostagmin Eeaction. — G-. Izar^^ and Ascoli call attention 
to a specific test which is named the meiostagmin reaction. Izar 
measures the size of the drop with a stalagmometer (an instru- 
ment for measuring the size of drops), and announces that the 
drop-forming property of various fluids becomes modified in cer- 
tain pathologic conditions. Izar has applied the test to syphilitic 
sera and found that the addition of the syphilis antigen increased 
the number of drops in the test fluid, while the serum of non- 
syphilitics had no such influence. 

Technic. — ^Use for the antigen an alcoholic extract of 
the spleen of a syphilitic fetus (0.5 gram of pulverized spleen 
mixed with 50 cubic centimeters of alcohol incubated for two 
hours, filtered and evaporated to 10 cubic centimeters, which 
was then diluted 1 to 100 parts with 0.85 per cent, salt solution) . 
The blood-serum is diluted 1 to 100 with similar solution. 
The number of drops formed by the diluted serum is determined 
before addition of the antigen. Then 1 cubic centimeter of the 
diluted serum is tested in the same way before adding the anti- 
gen. Then 1 cubic centimeter of the diluted antigen is added to 
9 cubic centimeters of the diluted serum, and the whole kept at 
37° C. (98.6° F.) for two hours, before the number of drops is 
again determined. There were always from 2 to 5 more drops 
in the fluid after addition of the antigen. Two investigations of 
serum from patients with leprosy gave no change in the number 

16 Munch, med. V^ochen., Jan. 25, 1910, Ivii, No. 4. 



464 SERODIAGNOSIS. 

of drops. This test is applicable to typhoid fever. Admixture 
of typhoid senim with an extract of typhoid bacilli increases the 
number of drops which the fluid is able to form. A positive 
specific reaction has also been obtained in tuberculosis. 

OPSONIC METHOD. 

Since the publication of Wright's original researches, 
Wright, Douglas, Eead, and many other laboratory and clinical 
workers have added much to our knowledge of individual resist- 
ance, the bacteriacidal power of the blood and other chemico- 
vital factors, so that many of the details of procedure insisted 
upon by Wright as necessary to a proper application of the 
method are no longer considered essential. Clinical practice 
demonstrates that in the employment of bacterial vaccines, the 
laboratory estimation of the opsonic index is rarely necessary as 
in most cases a perfectly satisfactory result may be obtained by 
what may be termed the "^^routine spacing-'' of the dose interval. 

For this reason and because of the technical difficulty sur- 
rounding an estimation of the opsonic index, the author believes 
it unnecessary to go into this technic in detail or to further 
discuss its use or value here. Current literature has many 
references to it, and a fair number of works on diagnostic and 
bacteriologic methods furnish a reliable guide for those desiring 
to employ it. 

THE SCHICK TEST FOR DETERMINING IMMUNITY 
TO DIPHTHERIA INFECTION. 

It is no longer necessary to give prophylactic injections of 
antidiphtheric serum to all those exposed or likely to be exposed 
to diphtheria, as it has been shown that many persons do not 
contract diphtheria even from what may be regarded as abun- 
dant exposure. The determination of this immunity dates 
from the work of B. S chick, ^"^ since which a large amount of 
clinical evidence has accumulated to demonstrate, without 
question, that the Schick test should be applied to all persons 
who have been exposed to diphtheria and that only those who 
react positively should be given prophylactic injections of 
antitoxin. 



17 Munch, med. Woch., Nov. 25, 1913, page 2608. 



THE SCHICK TEST. 465 

The technic of the test is most simple and the negative 
reaction, which indicates immunity to diphtheria, is not dif- 
ficult to determine. Some experience, however, is needed to 
separate the true positive reaction from certain atypical or so- 
called false, or pseudoreactions, especially at the end of the 
first 24 hours. Such a false reaction may occur in highly im- 
mune persons and is not always easily explained (see below). 
The test also assists in distinguishing cases which are diph- 
theria from those who are bacillus carriers. Diphtheria bacillus 
carriers usually develop relatively large amounts of antitoxin 
in the blood (and hence react negatively), whereas, in the acute 
stage of diphtheria, before injection of antitoxin the patient's 
blood contains little or no antitoxin, and the reaction is positive. 
In cases of acute diphtheria full doses of antitoxin always im- 
mediately modify or may completely inhibit the cutaneous 
reactions. IS 

Kolmer and Moshage^^ and others have summarized the 
relation of the skin reaction to the quantity of diphtheria anti- 
toxin present in the individual tested. They state that "per- 
sons reacting negatively to this test usually contain %o ^^^^ 
of diphtheria antitoxin per cubic centimeter of serum, and this 
amount of antitoxin is probably sufficient to protect against 
infection/' 

"Persons reacting weakly or strongly positively usually 
contain less than %o <^f a unit of antitoxin per cubic centimeter 
of serum or none at all. These persons may be regarded as 
susceptible to diphtheria and in the event of exposure to in- 
fection should be passively immunized.'^ These authors also 
find that in children from 1 to 15 years the preliminary use 
of the toxin test will eliminate the necessity of prophylactic 
doses of antitoxin in about 50% of cases. 

Pseudoreactions. — It has been found that in spite of the 
greatest care in performing this test, pseudoreactions occur, 
alike in susceptible and in immune persons. The difficulty in 
separating them is particularly evident during the first 24 hours 
after the injection, when it may be impossible to differentiate 



18 G. H. Weaver and L. K, Matier, Jour, of Infectious Diseases, March, 
1915, xvi. No. 2. 

19 American Jour, of Diseases of Children, March, 1915, ix. No. 3. 

30 



466 SERODIAONOSIS. 

the false from the true toxin reaction. On this account Kolmer 
and Moshage^o suggest that as a precautionary measure it is 
advisable to record all reactions, evidently non-traumatic, as 
positive unless a control bouillon injection is made at the same 
time. Delay in reading the reaction usually clears up the 
doubt. These investigators mention the following two common 
causes of pseudoreactions : — 

1. Traumatic, due to injection of fluid containing tri- 
cresol into the epidermis of persons whose skins are, for some 
reason, unduly sensitive. 

2. Local anaphylactic reactions of a general protein char- 
acter as described by Parke.^i 

Basis of Application of the Test. — In conducting the 
Schick toxin test for immunity to diphtheria it is advisable to 
employ a highly potent toxin and to inject about %o of i^he 
minimum lethal dose, so diluted with normal salt solution as to 
be contained in 0.05 to 0.1 c.c. The percentage of tricresol pres- 
ent should not exceed 0.25 per cent. The control fluid is com- 
posed of bouillon diluted to 10 or to 100 ; this should be injected 
at the same time in the same amount and greatly aids in detect- 
ing skin hypersensitivity and pseudoreactions. 

Traumatic Eeactions. — Clinical appearance — -Very small 
areas of erythema above the site of injection, measuring 2 or 3 
millimeters in the largest diameter, may safely be regarded as 
traumatic (Kolmer and Moshage). These reactions are usually 
recognized by their early appearance, their less circumscribed 
form, greater infiltration and the fact that they disappear in 
from 24 to 48 hours at the latest, while the spot is less pig- 
mented and superficial scaling is not noted.22 

Specificity. — Abraham Zingher^s reports /an examination 
of 1300 scarlet-fever cases of which 700 gave negative reactions. 
N"ot one of these negatives developed clinical diphtheria, al- 
though none were given immunizing doses of antitoxin and all 
were constantly exposed, in the wards, to cases of diphtheria. 



20 Jour. a. M. a., July 10, 1915, Ixv, No. 2. 

21 Archives Pediatrics, xxxi, 7, page 481, 1914, and in Proceedings N. Y. 
Pathological Society, Oct., 1914. 

22 Graeff and Ginsberg, Jour. A. M. A., April, 1915, Ixiv, 15, page 1205. 

23 Jour. A. M. A., July, 1915, Ixv, 4. 



THE SCHICK TEST. 4G7 

Technic. — A fine, sharp, but short pointed needle and an 
accurate syringe are necessary. A usual 1-c.c. "Eecord^' tuber- 
culin syringe with a platinoiridium needle is satisfactory. A 
standard concentrated diphtheria toxin is diluted 1 to 10 with 
0.5 per cent, phenol solution. This may be kept on ice for from 
one to two weeks. For use further dilutions are made in normal 
saline of such a strength that 0.1 c.c. contains %o niinimum 
lethal dose for a guinea-pig. This preparation is based on the 
following: 0.6 c.c. (minimum lethal dose) of toxin is diluted 
with 9.4 c.c. of the 0.5 per cent, solution of phenol; 1 c.c. of this 
dilution is again diluted with 99 c.c. of normal salt solution; 0.2 
c.c. of this represents %o minimum lethal dose. (See footnote 
8.) The usual site of injection is over the forearm, where, 
after having cleansed with alcohol, 0.2 c.c. of the secondary dilu- 
tion of the toxin is injected intracutaneously so that it pro- 
duces a small whitish weal in the skin. This is absolutely essen- 
tial to obtain a proper reaction. The reaction begins to appear 
within 24 hours and by from 36 to 48 hours the area of 
injection is grayish in color with considerable induration of 
the skin. There is usually a slight amount of pain. The size 
of the reaction is rarely less than that of a 10-cent piece. After 
a week the color begins to fade and as it does so there will be 
seen a small whitish center in the area of injection. This is 
followed by a slight brownish discoloration of the skin which 
persists for a week or ten days. With the development of the 
discoloration there is desquamation of the skin at the point of 
injection.24 

Koplick and Unger^s consider this method too cumbersome 
and advise the following technic. After the area of the skin 
on the forearm has been cleansed with alcohol, the latter is en- 
circled by the thumb and index finger and the skin held tensely 
between them. An ordinary hypodermic needle should be bent 
at a distance of ^ inch from its tip so as to make an angle 
of about 170 degrees. This is dipped into a bottle of pure un- 
diluted diphtheria toxin and then immediately inserted intra- 



24 Recently Parke and Zingher have improved the method and there may 
now be obtained in the market the toxin in capillary tubes each containing 
one 'minimal dose. 

25 Jour. A. M. A., April 1906, Ixvi, 16. 



468 SERODIAGNOSIS. 

dermatically. The angle of the needle aids this insertion and 
also determines the distance to which it is inserted into the skin. 
This needle is so constructed that when it is inserted its full 
length the amount of toxin carried in is approximately %o of 
the minimum lethal dose. They found that by weighing the 
needle before and after dipping, the difference was 0.0001 gm. 



XV. 
APPENDIX. 



DESCRIPTION OF OFFICE LABORATORY CABINET. 

Fig. 77 shows a semi-portable .laboratory cabinet containing 
reagents and apparatus sufficient to perform a majority of the 




Fig. 77.— Portable Clinical Laboratory Set, after Plan 
Suggested by the Author. (G. P. P. & Son Co.) 

simpler tests employed in the examination of uncertain sputum, 
gastric contents, and feces; also for the preparation of micro- 
scopic specimens of the blood and of bacteria. 

This may be obtained in the open market and will be found 
of great service to those desiring to follow clinical methods in 
connection with the study of their cases. It is finished in quar- 
tered oak. The dimensions are approximately 13 by 15 by 
26 inches. 

(469) 



470 APPENDIX. 



CLINICAL TERMS. 

Owing to the various and uncertain terms employed to 
designate the amounts of substances found, in clinical investiga- 
tions the following scheme has been successfully employed in 
our laboratory. 

Terms to be used in eixpressing the results of examinations 
of specimens in laboratory^ : — 





^ Questionable trace. 


Albumin, 


Very faint trace. 


Sugar, 


Faint trace. 


Indican, 


Trace. 


Acetone, - 


Strong trace. 


Bile, 


Moderate amount. 


Blood, 


Large amount. 


etc. 


Very large amount. 




Excessively large amount 


For Se 


>diments use: — 


Occasional 




Few. 




Moderate i 


lumber. 


Many. 




Very manj 




Excessively 


T large number. 



DISEASES IN WHICH LABORATORY TESTS ARE OF 
ESPECIAL VALUE.2 

The diseases are given in alphabetical order. The principal 
objects to be examined are enumerated; words in light-face 
capitals represent the most important laboratory findings. 
Words in black face represent tests which will usually require 
the services of a laboratory specialist. 



1 After Judson Daland. 

2 Modified from "Model Laboratory," by Henry Alberts, M.D., and Mildred 
E. Schuty, M.D., Jour. Iowa State Med. Soc, May, 1914. 



LABORATORY TESTS. 47I 

ACTINOMYCOSIS— 

1. Pus from abscess (or sputum ) —SULPHUR GRANULES OF 
THE RAY FUNGUS. 

AMEBIC DYSENTERY amebiasis. 

1. Feces and pus from abscesses— AMCEB A DYSENTERIC OR 
ENTAMOi^BA HISTOLYTICA. 

ANEMIA— 

1. Blood— ABSOLUTE AND DIFFERENTIAL LEUKOCYTE 
COUNT. 
—HEMOGLOBIN TEST. 
—BLOOD- PICTURE— microscopic. 

ANIMAL PARASITES— 

1. Blood — BLOOD-PICTURE — esp. eosinophilia and parasites. 

—HEMOGLOBIN. 

2. Feces— PARASITES OR OVA. 

—OCCULT BLOOD. 

ANTHRAX— 

1. Purulent discharge— BACTERIA. 

2. Blood-culture— BACTERIA. 

APPENDICITIS— 

L Blood— ABSOLUTE AND DIFFERENTIAL LEUKOCYTE 
COUNT. 

ASTHMA (bronchial). 

1. Blood-picture— EOSINOPHILIA. 

2. Sputum— EOSINOPHILIC LEUKOCYTES. 

— CURSCHMAN'S SPIRALS. 

— CHARCOT-LEYDEN CRYSTALS. 

BRAIN TUMORS 

or 
TUMOR-LIKE PROCESSES. 

1. Blood-picture— LYMPHOCYTOSIS. 

2. Cerebro-spinal fluid— NOGUCHI'S BUTYRIC ACID 1 

— NONNE'S. i. .... 

— LANGE'S COLLOIDAL GOLD, f ^^ syplnlis. 
TESTS (Positive Wassermann) j 
—CYTOLOGY. 

—TRACES OF BLOOD— in cerebral hem- 
orrhage. 
—SUGAR CONTENT. 

3. Urine— TRANSITORY GLYCOSURIA. 



472 APPENDIX. 

BRONCHIECTASIS. 

1. Sputum— TRISEDIMENTATION. 
—PUS CELLS. 
—FATTY ACID CRYSTALS. 

BRONCHITIS. Capillary. 

1. Sputum— MUCOID OR MUCOPURULENT. 

—PUS CELLS AND EOSINOPHILES. 
—BACTERIA. 

BRONCHO-PNEUMONIA. 

Same as capillary bronchitis. 

BUBONIC PLAGUE. 

1. Blood — Slow coagulation time. 

—ABSOLUTE AND DIFFERENTIAL LEUKOCYTE 

COUNT. 
—BACTERIA. 
CANCER. 

1. In general — Tissue examination. 

2. Of stomach. 

A— Gastric contents— ABSENCE OF FREE HYDROCHLORIC 
ACID. 

—INCREASE OF LACTIC ACID. 

—OCCULT BLOOD. 

—TISSUE FRAGMENTS IN WASHINGS. 
B— Feces— OCCULT BLOOD. 

3. Of kidney— HEMATURIA. 

4. Of pancreas— STEATORRHEA. 

5. Of uterus — Uterine scrapings. 

CHLOROSIS. 

1. Blood— ABSOLUTE AND DIFFERENTIAL COUNT. 
—HEMOGLOBIN. 

CHOLERA Asiatic. 
Feces — Bacteria. 

CONJUNCTIVITIS. 

1. Discharges— BACTERIA. 

CYSTITIS. 

1. Urine— PUS. 

—BACTERIA. 
—CHEMICAL ANALYSIS. 

DIABETES. 

1. Urine— SUGAR. 

—ACETONE. 

— Diacetic acid. 

— B-oxybutyric acid. 

2. Blood— BLOOD SUGAR. 

—ALKALINE RESUME OF PLASMA. 
—CARBON DIOXID TENSION. 



LABORATORY TESTS. 473 

DIPHTHERIA. 

1. Smear and Culture— BACTERIA. 

EMPHYSEMA. 

1. Blood— BLOOD-PICTURE— HYPEREOSINOPHILIA. 

ENDOCARDITIS. 

1. Blood— LEUKOCYTE COUNT. 
—BLOOD CULTURE. 

FILARIASIS. 

1. Blood— PARASITES. 

—DIFFERENTIAL COUNT. 

GLANDERS. 

1. Discharge from nose and lesions — Bacteria. 

GONORRHEA. 

1. Discharge— BACTERIA. 

2. Urine— PUS AND SHREDS. 

HEART DISEASE— Chronic Valvular. 

1. Sputum— HEART LESION CELLS. 

HYPERACIDITY OF STOMACH— Gastrosuccorrhoea acida. ' 
1. Stomach contents— HYPERCHLORHYDRIA. 

— TRISEDIMENTATION OF VOMITUS. 

HYDATID DISEASE. 

1. Fluid from cyst, sputum, etc.— HOOKLETS. 

INFLUENZA. 

1. Sputum— BACTERIA. 

INTESTINAL HELMINTHIASIS— See Animal Parasites. 

LEAD POISONING. 

1. Blood — Blood-picture — Basophilic granular degeneration. 

LEPROSY. 

1. Blood from nodules — Bacteria. 

LEUKEMIA. 

1. Blood— ABSOLUTE AND DIFFERENTIAL LEUKOCYTE 
COUNT. 

LUNG ABSCESS AND GANGRENE. 
1. Sputum— PUS CELLS. 

—ELASTIC TISSUE. 
—FRAGMENTS OF LUNG. 
—BACTERIA. 
IVIALARIA. 

1. Blood— SPECIAL STAIN FOR PLASMODIUINI. 



474 APPENDIX. 

MEASLES. 

1. Blood— DIFFERENTIAL LEUKOCYTE COUNT. 

MENINGITIS. 

1. Blood— ABSOLUTE AND DIFFERENTIAL LEUKOCYTE 

COUNT. 

2. Cerebro-spinal fluid— BACTERIA. 

—DIFFERENTIAL CELL COUNT. 
—GLOBULIN CONTENT. 
—SUGAR CONTENT. 
MYELOMATOSIS. 

1. Urine— BENCE JONES'S PROTEIN. 

NEPHRITIS. 

1. Urine— CHEMICAL EXAMINATION— ALBUMIN. 

—MICROSCOPIC EXAMINATION— CASTS, etc. 

2. Functional Tests. 

— phenolsulphonphthalein. 

— ph concentration. 

— co2 tension of alveolar air. 

3. Blood-pressure — 

OTITIS MEDIA AND MASTOIDITIS. 

1. Discharge— BACTERIOLOGIC EXAMINATION. 

PARESIS. 

1. Blood — ^Wassermann. 

2. Cerebro-spinal fluid— ABSOLUTE AND DIFFERENTIAL LEU- 

KOCYTE COUNT. 
— NOGUCHI'S TEST. 
— LANGE'S COLLOIDAL GOLD TEST. 
— NONNE'S TEST. 
PNEUMONIA. 

1. Sputum— BACTERIA. 

—TYPING OF BACTERIA. 

2. Blood— DIFFERENTIAL LEUKOCYTE COUNT. 

3. Urine— CHLORIDES. 

PSEUDOLEUKEMIA— Hodgkin's disease. 

1. Blood— ABSOLUTE AND DIFFERENTIAL LEUKOCYTE 

COUNT. 

2. Culture of fluid from gland. 

PYELITIS. 

1. Urine— PUS. 

—BACTERIA. 
—OCCULT BLOOD. 

— Permeation test. 

RABIES. 

1. Brain tissue — Negri bodies. 



LABORATORY TESTS. 475 

SCARLATINA. 

1. Blood— DIFFERENTIAL LEUKOCYTE COUNT. 

SEPTIC INFECTIONS. 

1. Blood— ABSOLUTE AND DIFFERENTIAL LEUKOCYTE 

COUNT. 
— Bacteriologic examination. 

2. Pus— BACTERIA. 

SYPHILIS. 

1. Primary lesion — ^Smear for spirochetae. 

f Blood — Wassermann. 
„ ^ ^ - . — HECHT-WEINBERG. 

2. Secondary lesion -. ^ , . . r, • -, 
3 Tertiarv— I .!' Cerebro-spmal fluid. 

3. ;^ertiary _<! -NOGUCHI'S TEST. 

4. Tabes and G. P. I. J | —LANGE'S TEST. 

(^ — NONNE'S TEST. 

TETANUS. 

1. Discharge— Smear for specific bacteria. 

TONSILLITIS. 

1. Exudate— BACTERIOLOGIC EXAMINATION. 

2. Blood— DIFFERENTIAL LEUKOCYTE COUNT. 

TRICHINOSIS. 

1. Blood— HYPEREOSINOPHILIA. 

2. Feces— PARASITES Adults or embryos. 

3. Muscle tissue — Trichinella embryos encysted. 

TRYPANOSOMIASIS. 

1. Blood— DIFFERENTIAL LEUKOCYTE COUNT. 

— TRYPANOSOMES. 

2. Cerebro-spinal fluid— TRYPANOSOMES. 

TUBERCULOSIS. 

1. Sputum- TUBERCLE BACILLI. 

2. Blood— LYIVIPHOCYTOSIS. 

3. Tissue — Tubercles. 

4. Reaction— TUBERCULIN. 

TYPHOID FEVER. 

L Blood— WIDAL— MACROSCOPIC. 

— Widal — Microscopic. 
— Bacteria. 

TUIMORS- in general. 

1. Tissue — Microscopic examination. 

VINCENT'S ANGINA. 

1. Exudate — Bacteriologic examination. 



476 



APPENDIX. 



URINALYSIS. 

Report of Clinical Laboratory. 



Physical Characteristics. 



Quantity in ") 
24 hours. J 
Color, 

Appearance, 
Odor, 
Sediment : — 

Mucus, 

Urates, 

Phosphates, 

Uric Acid, 

Pus, 
Specific Gravity, 
Reaction, 



c.em. 
oz. 



Quantitative DETERMINATIo^- s. 

'Heat, 10^ Acetic Acid. 
Albumin -i Heller's Nitric Acid. 
I^Heat and Nitric Acid. 

r Fehling's. 
Glucose J Phenylhydrazin. 

LBottger's Bismuth. 

Indican, 

Skatol, 

Acetone, 

Bile Pigments, 

Blood, 

Diazo, 

Aceto-acetic Acid, 

B-Oxybutyric Acid, 

Drug Reactions, 

Cammidge Reaction, 



Quantitative Determination. 



Albumin (Esbach), (Purdy), 

Glucose (Fehling's), (Fermentation), 

Urea, 

Ethereal Sulphates, 

Chlorids, 



Microscopic Examination, 



(Centrifugated), 



Casts.- 



'■ Hyaline, 
Granular, 
Fatty, 
Epithelial, 
Blood, 
Waxy, 
Bacterial, 

Cylindroids, 

Mucus, 



(SedimentedV 



Erythrocytes, 
Leukocytes, 
Pus cells, 
Epithelia, 
Spermatazoa, 
Bacteria, 
Yeast Spores, 
Trichomonides. 



Voided, A.M P.M. 



Examined, A.M. . 



..P.M 



Examined by 



DILUTING SOLUTIONS FOR BLOOD CELL COUNT. 477 

METHODS FOR FIXING BLOOD AND OTHER FILMS UPON 
GLASS SLIDES PREPARATORY TO STAINING. 

1. Equal parts of absolute alcohol and ether. The specimen 
should be immersed in this for half to two hours. This method 
is particularly good for malarial parasites and degeneration in 
blood-cells. 

2. Absolute alcohol for five minutes. 

3. Flemming's solution: — 

Chromic acid, 1 per cent 15 parts. 

Osmic acid, 2 per cent 4 parts. 

Glacial acetic acid 1 part. 

The blood-specimens, as soon as they are made, before they 
have time to air-dry, are plunged into this solution and allowed 
to remain for ten minutes. They are then washed in running 
water for ten minutes and dried. This solution is very useful 
for demonstrating the chromatin of nuclei. 

4. Vapor of formaldehyde. 

5. Heat. 

6. Methyl alcohol. This is coming rapidly in favor, as it 
can be mixed with the stain, thus reducing the time of preparing 
specimens. 

DILUTING SOLUTIONS FOR BLOOD CELL COUNT. 

A. E. Osmond recommends the following diluting solutions 
for the white and red count as being as efficient as the more 
elaborate diluting solutions, while having the advantage that 
they do not require a chemist for their preparation. "These 
solutions may be made offhand.^' 

For counting the red cells, normal salt solution tinged with 
a drop of eosin. 

For the ivhite cells a 1 per cent, solution of acetic acid 
tinged with a drop of methylene-blue. He states that a yellow 
artificial light shows the cells up best, and the only real need 
for the coloring in these solutions is to distinguish one from 
the other. 

Red Cell Count. — Two and one-half per cent, solution 
potassium bichromate in distilled water. This preserves the 
red cells and destroys the white cells. The solution is perma- 
nent, but may have to be filtered occasionally to remove sediment. 



478 APPENDIX. 

White Cell Count. — One-half per cent, solution of acetic 
acid. This solution hemolizes the red cells and accentuates the 
structure of the white cells. It is stable, but may develop a mold, 
which may be removed by filtering. 

Piteield's Solution eor White Cells. — ^Acacia gum, 
20 grams; distilled water, 500 cubic centimeters; mix, dissolve, 
and add glacial acetic acid, 50 cubic centimeters, gentian violet, 
1 decigram; mix, warm, and filter while warm through a wet 
filter. This makes a superior diluting fluid for counting leuko- 
cytes, because it is viscid and does not flow out of pipettes easily. 
Thus, these can be filled more accurately because the fluid flows 
much more slowly. Neat drops of definite size may be slowly 
put up on the counting chamber. The pipette, filled, may be 
carried without fear of spilling the contents. The leukocytes 
do not settle in the mixing chamber quickly and remain evenly 
distributed throughout the fluid. The end of the pipette must 
be wet before the fluid can be blown out, as the end of the 
pipette is quickly sealed with the acacia. 

Diluting Solution for Counting Blood-Cells. Hayem's 
Solution for Counting Erythrocytes: — 

Mercuric bichlorid 0.5 gm. 

Sodium sulphate 5.0 gm. 

Sodium chlorid 1.0 gm. 

Aq. dest 200.0 c.c. 

DILUTING FLUID FOR BLOOD PLATELET COUNT.3 

Solution I : — 

"Brilliant cresyl blue" 1.0 gm. 

Distilled water , , 100.0 c.c. 

Solution should be kept in cool place to prevent formation 
of mold. 
Solution II : — 

Potassium cyanide 1.0 gm. 

Distilled water 1400.0 c.c. 

To use: — 

Mx 1 part solution I vnth 3 parts solution II, filter and 

use immediately. 

3 W^right and Kinnicutt : Jour. Am. Med. Assn., Ivi, 1455, 1911. 



STAINS. 479 

STAINS. 

Carbol Gentian- Violet^ : — 

Gentian-violet, cone, alcoholic sol 10.0 c.c. 

Carbolic acid, 5 per cent, watery sol. . . 100.0 c.c. 

Chezinsky Staik^ : — 

Methylene-blue, sat. aq. sol 45.0 c.c. 

Eosin, 0.5 per cent in 70 per cent, alcohol . ... 25.0 c.c. 

Distilled water 45.0 c.c. 

The specimens are fixed in absolute alcohol for from five 
to thirty minutes, and stained in a thermostat at 37° C. for 
from three to six hours. 

Czaplewskt's Stain^. — One gram of fuchsin is dissolved 
in 5 cubic centimeters of liquid carbolic acid in a dish; 50 cubic 
centimeters of glycerin are then added, stirring constantly, and 
finally diluted with water to 100 cubic centimeters. The solution 
is said to keep extremely well, and does not need to be filtered. 

Ebner's Fluid'^ : — 

Hydrochloric acid 2.5 c.c. 

Sodium chlorid, C. P 2.5 c.c. 

Distilled water 100.0 c.c. 

Alcohol, 95 per cent. ....... 500.0 c.c. 

Ehelich's ''Triacid" Stain^ : — 

Orange G 13.0-14.0 c.c. 

Acid fuchsin 6.0- 7.0 c.c. 

Distilled water ., 15.0 c.c. 

Methyl green 35.5 c.c. 

Alcohol 10.0 c.c. 

Glycerin 10.0 c.c. 

The three stains, orange G, acid fuchsin, and methyl green, 
are prepared in saturated aqueous solutions, and then mixed in 
the above amounts while being shaken thoroughly. 

4 Gram's method, p. 433. 

5 For differential blood count, p. 68. 

6 For tubercle bacillus, p. 33. 

^ Decolorizing fluid for tubercle bacillus, p. 33. 
8 Differential blood count, pp. 68 and 174. 



480 APPENDIX. 

EosiN AND Hematoxylin (Delafield's)^. — Two solutions 
are required with equally good results, the eosin solution, men- 
tioned below, and hematoxylin, the formula for which is: — 

Hematoxylin crystals 4.0 gm. 

Alcohol (absolute) 25.0 c.c. 

Ammonium-alum crystals C. P 52.0 gm. 

Distilled water 400.0 c.c. 

Glycerin C. P . 100.0 c.c. 

Methyl alcohol, C. P ,. . 100.0 c.c. 

and which is prepared as follows : Eub the hematoxylin crystals 
up with the alcohol until they are dissolved; then place the 
solution in a loosely corked glass bottle, allowing it to stand 
exposed to the light for four days. Dissolve the ammonium-alum 
in the water and allow it to stand exposed in the same way for 
four days. At the end of this time mix the two solutions, 
shake thoroughly, and filter at the end of three hours. Add 
the glycerin and methyl alcohol to the filtrate and allow this 
to stand overnight. Filter the mixture, place it in a clear 
bottle, and allow it to ripen, exposed to the light for six weeks, 
when it is ready for use. This stain is applied and allowed 
to act for from two to four minutes after the eosin stain has 
been thoroughly washed from the slide. 

Eosin and Methylene-BlueI^: — 

Eosin 0.5 per cent, in 70 per cent, alcohol. 

Stain for a few minutes, wash, blot, and apply. 

Methylene-blue 1 per cent, aqueous. 

Stain for a few minutes. 

Gabbott^s Method! 1: — 

A — Fuchsin 1.0 gm. 

Absolute alcohol 10.0 c.c. 

Carbolic acid (5 per cent.) 100.0 c.c. 

B — Methylene-blue ... 2.0 gm. 

Sulphuric acid (35 per cent, sol.) . . . 100.0 c.c. 



9 Differential blood count, p. 70. 

10 Differential blood count, pp. 68, 70 and 167. 

11 For tuberce bacilli, p. 33. 



STAINS. 481 

GiEMSA Improved Stain^^ ; — 

Azur II eosin ., , 3.0 gm. 

Azur II ., 0.8 gm,. 

Exsiccate, pulverize, and sift. 
Dissolve in chemically pure glycerin at 

60° C 250.0 e.c. 

When solution is complete add: — 
Methyl alcohol at temperature 60° C. . 250.0 c.c. 
Shake well, allow to stand for twenty-four hours, then 
filter. 

Geam^s Iodine! ^ : — 

lodin 1.0 gm. 

Potassium iodid 2.0 gm. 

Distilled water 300.0 c.c. 

Grunthal's Stain14 .__ 

Kresylecht violet 0.07 gm. 

Methylene blue 0.10 gm. 

Glacial acetic acid 3.00 c.c. 

Water 1000.00 c.c. 

Keeps 4 months or more. 

Hematoxylin-Eosin (Ehrlich^s mixture)!^: — 

Eosin (crystals) 0.5 gram. 

Hematoxylin 2.0 grams. 

Absolute alcohol, 

Distilled water, I of each 100.0 c.c. 

Glycerin, 

Glacial acetic acid 10.0 c.c. 

Alum in excess. 

This mixture should be allowed to stand for several weeks 
before it is ready for use. The specimens are stained in from 
half to two hours. 



12 For Treponema pallidum, p. 173. 

13 Gram's differential method, pp. 39 and 433. 

14 For bacillus diphtherise, p. 428. 

15 Differential blood count p. 70. 

31 



482 APPENDIX. 

Kooh-Ehrlich Gentian-YioletI^ : — 

Take distilled water, 100 cubic centimeters, and add anilin 
oil, drop by drop, until the solution has an opalescent appear- 
ance. The vessel containing the mixture should be thoroughly 
shaken after the addition of each drop. It is then filtered 
through moistened filter paper until the filtrate is clear. To 
100 cubic centimeters of the filtrate add 10 cubic centimeters of 
absolute alcohol and 2 cubic centimeters of concentrated solution 
of gentian-violet. 

LoFFLEPt's Alkaline Methylene-blueI^ : — 

Concent, alcohol solution methylene-blue. . 30.0 c.c. 
Potassium hydrate (%oooo) 100.0 c.c. 

Pappenheim's Solutionis : — 

Corallin 1 part. 

Absolute alcohol , 100 parts. 

Add to the above solution methylene-blue in bulk to sat- 
uration. Finally add 20 parts of glycerin. 

Reagents for Staining FlagbllaI^ : — 
Mordant. 

Tannic acid (20 ac. to 80 water) 10.0 c.c. 

Ferric sulphate cold sat. sol 5.0 c.c. 

Fuchsin sat. watery sol 1.0 c.c. 

Adjuvants. 

Sodium hydrate 1 per cent, aqueous solution. 

Sulphuric acid (1 c.c. equal 1 c.c. of 1 per cent.. NaOH). 

Unna's Orcein Stain^O; — 

Orcein in substance 1.0 gm. 

Hydrochloric acid , 1.0 c.c. 

Absolute alcohol . 100.0 c.c. 



16 For capsule staining, pp. 40 and 435. 

17 Counter-stain for tubercle bacillus, p. 35. 

18 For tubercle bacillus, pp. 33 and 263. 

19 For staining flagella, p. 434. 

20 For elastic tissue in sputum, p. 28. 



STAINS. 483 

ZiEHL^s Carbol-Fuchsin^i : — 

Fuchsin in substance 1.0 gm. 

Carbolic acid (cryst.) 5.0 gm. 

Alcohol (95 per cent.) 10.0 c.c. 

Distilled water 100.0 c.c. 

Or it may be prepared by adding to a 5 per cent, watery 
solution of carbolic acid a saturated alcoholic solution of 
fuchsin until a metallic luster appears on the surface of the 
liquid. 

Koch-Ehelich Analin Water Fuchsin22: — 

Prepare saturated analin water by placing 2 c.c. of analin 
oil in a test-tube; add 15 c.c. of distilled water and shake 
vigorously. Filter into a small porcelain dish and add a satu- 
rated alcoholic solution of fuchsin^ drop by drop with stirring, 
until a metallic sheen appears on the surface of the mixture. 
This stain decomposes very readily and should be prepared 
fresh for each examination. 

Toisson's Solution for Simultaneously Diluting Eed and 
White Cells: — 

Methyl violet . 0.05 gm. 

ISTeutral glycerin . . 30.00 c.c. 

Aq. dest 80.00 c.c. 

Mix and add: — 

Sodium chlorid 1.00 gm. 

Sodium sulphate 8.00 gm. 

Aq. dest 80.00 c.c. 

Filter. Twelve minutes required to stain white blood- 
cells. 

A New and Stable Solution of Gentian-Violet^s -. — 

The decided tendency of the average gentian-violet solution 
to decompose, especially in warm weather, is a difficulty fre- 
quently encountered by the laboratory worker. The result is; 
loss of the entire solution and much time required to make a new 

-1 For tubercle bacillus, p. 32. 
-- For staining flagella, p. 434. 
-3 For staining capsules, p. 40. 



484 APPENDIX. 

one. The following suggestions are practical and obviate this 
difficulty. 

Dr. E. Burvill-Holmes has had success with the addition of 
3 to 5 per cent, of glycerin to the stain which improves its 
stability if kept in a dark, cool place. Muir and Eitchie recom- 
mend the use of phenol water 1 part in 10. These methods 
while being improvements do not prevent decomposition of the 
stain. Eobert Kilduffe recommends the following preparation 
which he has found to work admirably. Two stock solutions 
are employed: A. 5 cubic centimeters 40 per cent, formalde- 
hyde are added to 95 cubic centimeters of distilled water. B. 
Saturated alcoholic solution of gentian-violet. Mixing these in 
the proportion of 25 parts of B and 75 parts of A such a solu- 
tion has been kept for a long time at ordinary temperatures with- 
out deterioration. The advantages of this solution are said to 
be: (1) It does not decompose. (2) Moulds cannot grow in it. 
(3) No modification of technic necessary. (4) Preparations 
made with it are sterile. , 

Universal Staining Method^^ : — 

The discovery of the process whereby two or more colors 
could be chemically combined in one staining reagent, marked 
a great advance in the field of hematology. It affords greater 
opportunity for more detailed study of the minute structure of 
the cells of the blood, which in turn has resulted in a better 
classification of the different elements through the differentia- 
tion of new and distinct varieties which, until recently, have 
been unrecognized. Of these combination stains the Polychrome 
or "universaP' staining method is by far the best and most prac- 
tical, and is now rapidly superseding the older and more cum- 
bersome methods. For this reason it becomes a matter of con- 
siderable importance, almost a necessity, that the clinical worker 
should have at hand one or more of these stains ready for imme- 
diate use when occasion requires. 

Unfortunately the preparation of this class of stain in- 
volves considerable expenditure of time, and demands no small 
amount of chemical knowledge and manipulative skill. These 



24 For blood corpuscle differentiation, p. 68. 



STAINS. 485 

factors combine to limit the preparation of these stains to a 
comparatively small number of experienced workers, while the 
majority of the profession is dependent upon unstable liquid 
stains^ obtainable through the supply houses, the composition 
of which is frequently so variable as to render the results value- 
less. Further, all aniline stains of this character are prone to 
decomposition when kept even for a moderately long time in 
solution. 

Fortunately the demand for a uniform and reliable stain 
has recently been met by a London Pharmaceutical House,^^ who 
now carry in stock a number of very uniform and perfectly re- 
liable stains under the name of "Soloid" brand. These stains 
are very carefully prepared and their composition practically 
uniform. A definite quantity of the dried stain is compressed 
into a tiny tablet and dispensed in vials containing six. Each 
package is accompanied with information indicating the proper 
dilution and best working conditions for that particular stain. 

The "Soloid^^ stains permit of the preparation of small 
quantities of liquid stain whereby waste through evaporation or 
decomposition is reduced to a minimum; at the same time the 
results obtained, as far as the author has employed them, are in 
every way satisfactory. 

Practically all the stains in common use, as well as the 
rare ones, are now prepared by this manufacturer, together with 
appropriate mordants, decolorizers, etc. These may be obtained 
of any reliable drug house or instrument dealer. 

The Polychrome MBTHYLBN-EhBLUE-EosiN Stains^^ (Ro- 
mano wski). — There are about fifteen different modifications of 
this stain. The majority of them are difficult of manufacture, 
even by an expert, so it is recommended that they be bought 
ready-made from the laboratory apparatus and supply houses 
which make them. 

For those who desire to make this stain for themselves, the 
Wright stain and a modification by Hastings, ^"^ are appended, 
the two having the greatest popularity. 

25 Burroughs Wellcome & Co., with a branch at 45 Lafayette Street. New- 
York City. 

26 iTor differential blood count, p. 68. 

27 Hastings : Johns Hopkins Hospital Bulletin, 1905. 



486 APPENDIX. 

All the Eomanowski stains are made with wood alcohol, 
which, during the first portion of its application, acts as a 
fixative. 

HASTiiirGS^s Stain". 2 8 — The dry stains necessary are eosin 
(water solution), yellow (Gruebler), and methylene-blne (Ehr- 
lich's rectif.) (Gruebler). 

Solution "A!' — Eosin 1 per cent, aqueous. 
Solution "B^^ — Alkaline methylene-blue 1 per cent. 

aqueous. 
Solution ^^C^^ — Methylene-blue 1 per cent, aqueous. 

Solution "A*' may be kept ready-made, solutions ^^B" and 
"C" must be made fresh. 

To prepare solution "B," use a warm 1 per cent, solution 
of dry powdered sodium carbonate. Add to it one per cent, of 
methylene-blue powder, and heat over a water bath for 15 
minutes. Add 30 cubic centimeters of water for each 100 cubic 
centimeters of the original fiuid, and heat again for 15 minutes. 
Then pour off the solution from the residue and divide) into 
two equal parts. To one part add enough 12.5 per cent, acetic 
acid solution to make a faintly acid reaction. This is best 
determined by taking a piece of blue litmus paper and allowing 
a drop to fall upon it, taking as the end reaction the point at 
which the margin of the drop after absorption in the paper 
shows a faint pink. Then add the remaining unneutralized por- 
tion to this. 

To mix the stain use distilled water, 1000 cubic centimeters ; 
Solution "K" 100 cubic centimeters; solution "B," 200 cubic 
centimeters; solution "C," 70 to 80 cubic centimeters. In add- 
ing solution "C" put in 70 cubic centimeters at once, and stir 
well ; if no precipitate appears, add 1 cubic centimeter at a time 
until one does appear. After the precipitate appears the stain 
is allowed to stand for half an hour and then filtered through 
one filter. Forced filtration is generally necessary. 

The dry residue is removed from the paper and pulverized. 
It may be kept in this form or dissolved in Merck's pure methyl 
alcohol. Seven- to nine- tenths of a gram of dried stain is 
usually obtained. Three-tenths of a grain is dissolved in 100 

28 For blood cells and malarial parasites, pp. 68 and 161. 



STAINS. 487 

cubic centimeters of alcohol for the staining solution. In dis- 
solving the stain it must be rubbed up with the alcohol in a 
mortar, as the powder is with difficulty soluble. 

If there are more than nine-tenths gram of the dried stain 
obtained, the preparation is useless, and its preparation should 
be begun again. 

For each new lot of stain made up, one must determine 
the relative proportion of stain and water used in staining, and 
the relative lengths of time during which the pure and diluted 
stain is allowed to act. 

Usually 2 drops of stain on the smear for one minute and 
then 4 drops of water added and allowed to act for four minutes 
gives the best result. 

For uniformity in dropping a dropper should be used. 

Wright^s Stain.29 — To a 0.5 per cent aqueous solution 
of sodium bicarbonate add methylene-blue (B. X. or "medici- 
nally pure") in the proportion of 1 gram of the dye to each 
100 cubic centimeters of the solution. Heat the mixture in a 
steam sterilizer at 100° C. for one full hour, counting the time 
after the sterilizer has become thoroughly heated. The mixture 
is to be contained in a flask or flasks of such a size and shape 
that it forms a layer not more than 6 centimeters deep. After 
heating, allow the mixture to cool, placing the flask in cold 
water if desired, and then filter it to remove the precipitate 
which has formed in it. It should, when cold, have a deep 
purple-red color when viewed in a thin layer by transmitted 
yellowish artificial light. It does not show this color while 
it is warm. To each 100 cubic centimeters of the filtered mix- 
ture add 500 cubic centimeters of a 0.1 per cent, aqueous solu- 
tion of "yellowish water soluble" eosin and mix thoroughly. Col- 
lect the abundant precipitate, which immediately appears^ on 
a filter. When the precipitate is dry, dissolve it in methyl 
alcohol (Merck's reagent) in the proportion of 0.1 gram to 60 
cubic centimeters of the alcohol. In order to facilitate solution, 
the precipitate is to be rubbed up with the alcohol in a porcelain 
dish or mortar with a spatula or pestle. This alcoholic solution 



29 For differential blood count, p. 68, 



488 APPENDIX. 

of the precipitate is the staining fluid. It should be kept in 
a well-stoppered bottle because of the volatility of the alcohol.^® 
All polychrome methylene-blue stains require experiment, 
since different mixtures by the same method require slight 
variations in their use. These must be ascertained by trial. 
Use distilled water to wash the specimen, since tap-water may 
ruin it. 

Labeling Smears: — 

As it often occurs that a number of slides are made at one 
time, or a number of slides from one patient are taken at dif- 
ferent hours, it is necessary that such slides should be labeled 
at once. The most convenient method is that of writing on the 
end or back of a slide with ordinary ink. This should be quite 
dry before the staining process is begun, then there will be no 
fear of it washing off. Another method suggested by Powell is : 
After making a dry film, the name, date, and other necessary 
information are scratched on the film, with the head or point of 
a needle, the film used being so extensive that the writing in no 
way interferes with subsequent study. In place of the needle, 
E. H. von Ezdorf suggests the use of an ordinary black lead 
pencil, preferably soft. The label thus made on the blood fibn 
being a carbon deposit remains permanent and is not affected 
by the staining and washing of the slide. 

Substances to Prevent Foaming During Aeration 
Processes : — 

Caprylic alcohol Benzol 

Amylic alcohol Kerosene 

Toluene 

MtJIR^S MORDANT^l : 

Corrosive sublimate, sat. aq. sol., 2.0 c.c. 

Tannic acid, 20 per cent. aq. sol., 2.0 c.c. 

Potash alum, saturated aq. sol., 5.0 c.c. 



30 Jour. Am. Med. Assn., vol. Iv, 1910, p. 1979. 

31 Muir's method for staining capsules, p. 436. 



NORMAL, DECINORMAIi AND STANDARD SOLUTIONS. 489 

NORMAL, DECINORMAL AND STANDARD SOLUTIONS. 

A normal solution (^) in substance is one which contains 
the hydrogen equivalent of its molecular weight dissolved in 
a liter of water, hydrogen being considered for analytical 
purposes as 1. 

Equal volumes of normal solutions should combine exactly 
with each other; for example, 1 cubic centimeter of ~- hydro- 
chloric acid should exactly neutralize 1 cubic centimeter of ^ 
sodium or potassium hydrate or 10 cubic centimeters of -^ 
alkali. 

A decinormal (~) solution is a solution of such a 
strength that 10 cubic centimeters of alkali will exactly neutral- 
ize 1 cubic centimeter of a -y- solution of acid. Decinormal 
solutions are usually prepared by diluting a ^ solution with 
9 parts of distilled water ; then if absolute accuracy is demanded 
it should be titrated and standardized with a normal solution 
of authentic accuracy. 

A standard solution is one having an arbitrary amount of 
a chemical substance in solution, this amount being usually 
determined by convention, or arranged for convenience in 
performing certain tests. 

These solutions may be obtained accurately standardized 
from reliable manufacturers. 

A molecular solution (mol.) is prepared by dissolving the 
full molecular weight of a substance, without regard to its 
hydrogen equivalent, in 1 liter of water. From such a solution 
further dilutions may be prepared by appropriate dilutions. In 
determining the hydrogen-ion concentration of the blood plasma 
two %5 mol. solutions are employed and are prepared as follows : 

Potassium di-hydrogen phosphate ..... 9.078 gm. 

Distilled water 1000.000 c.c. 

Di-sodium hydrogen phosphate (desic.) 11.876 gm. 
Distilled water ,. . . . . . 1000.000 c.c. 



490 APPENDIX. 

Table of Chemicals Frequently Used in Normal Solution — 

Together with the Amount in Grams Required 

TO Prepare JSuch Solution. 

Oxalic acid C2O4H2,2H20 . . . 63.024 grams per liter 

Hydrochloric acid HCl 36.46 " " " 

HoSO. 
Sulphuric acid — - 49.04 " " " 

Sodium hydrate NaOH 40.058 " " " 

Potassium hydrate KOH 56.16 " " " 

Ammonium hydrate NHg 17.064 " " " 

Sodium carbonate t t 53.05 " " " 

2 

^., ., , (AgNOs 169.97 " " " 

Silver nitrate < ° , ^„ „^ 

(Ag 107.92 " " " 

rAgNOo 

9Q Ofi " " " 

Silver nitratei ^ s^gs 

LAg 18.499 " " " 

Sodium chloride NaCl 58.5 " " 

KMn04 
Potassium permanganate ... 31.63 " " " 

Decinormal Sodium Hydrate. — A normal sodium hydrate 
solution is one which contains in each liter as many grams of the 
substance in question as its equivalent weight. A decinormal ^ 
solution is made from this by diluting 9 times; because of the 
facility with which this substance^ takes up water ^ and so is con- 
stantly changing in weight, it is best to start with a normal 
oxalic acid solution as a basis. 

The molecular weight of oxalic acid is 126.024 and, as the 
acid is dibasic, a normal solution would contain one-half of 
the molecular weight; that is, 63.012 grams. For ordinary work 
the commercial acid may be assumed as sufficiently pure to use as 
standard. Sixty-three grams of a well-crystallized and chemically 
pure oxalic acid are dissolved in distilled water and the volume 
made up to exactly 1 liter. Now, a normal sodium hydroxid 
solution will require, in order to neutralize a given volume of the 
normal oxalic acid solution, exactly the same volume. A few 
drops of alcoholic phenolphthalein solution are added as an 
indicator to 10 cubic centimeters of normal oxalic acid solution. 



NORMAL, DECINORMAL AND STANDARD SOLUTIONS. 49 1 

Then to this an approximately normal sodium hydroxid solution 
(40 grams dissolved in 900 cubic centimeters water) is added 
from a buret until the acid is neutralized — i.e.^ until the mixture 
takes on a permanent reddish color. If the normal NaOH solu- 
tion is correct, it will require exactly 10 cubic centimeters. 
Generally speaking, less is needed. For instance, if we employed 
9.5 cubic centimeters of the ^ NaOH for neutralization, we 
must add 0.5 cubic centimeter of water to each 9.5 cubic centi- 
meters of the solution. Then 10 cubic centimeters of the 
normal NaOH solution will correspond to exactly 10 cubic 
centimeters of the normal oxalic acid solution. From this we 
can easily estimate how much water to add to the 1000 cubic 
centimeters of solution — i.e., ~ X 0.5. Normal sodium 
hydroxid solution is too strong to use for the titration of the 
gastric juice; hence we prepare ^ by diluting the normal 
solution with 9 parts distilled water. 

Decinormal Hydrochloric Acid Solution. — This solu- 
tion is prepared by diluting 15 cubic centimeters of C. P. 
hydrochloric acid up to 1000 cubic centimeters with distilled 
water; 10 cubic centimeters of this is then titrated with the 
standardized ^ solution of sodium hydrate and corrected 
according to the method outlined above. 

Sodium Tiiiosulphate Standard (K/IO) Solution.^^ — 
Weigh out 25 grams of ordinary c. p. sodium thiosulphate or 
24.83 grams of the pure dry recrystallized salt. Dissolve in 
water and dilute to a liter. Boiled distilled water must be used. 
Keep in a bottle with a siphon arranged and carrying a soda 
lime tube to exclude CO2. 

It is best standarized against acid potassium iodate 
KH(I03)2. Weigh out accurately 0.3249 gram of acid potas- 
sium iodate. Dissolve in 50-cubic centimeter flask, rinsing the 
beaker carefully, and make to mark with water. This solution 
is exactly decinormal. Pipette out 25 cubic centimeters into an 
Erlenmeyer flask, add 1 gram of potassium iodide dissolved in 
a little water, and a few cubic centimeters of dilute hydrochloric 
acid. Titrate immediately with the thiosulphate solution. 



32 DetGrmination of acetone and acetoacetic acid, p. 533. 



492 APPENDIX. 

When the sohition becomes pale yellow add a few cubic centim- 
eters of 1 per cent, solution of soluble starch and titrate to loss 
of blue color. 

Iodine Solution (N/IO).^^ — Weigh out 12.685 grams of 
pure resublimed iodine into a small weighing bottle using a 
porcelain spatula. Dissolve 18 grams of pure KI in about 150 
c.c. of water. Transfer the iodine to a liter flask washing out 
the last traces with some of the KI solution, which is then 
poured into the flask. Stopper and shake occasionally until 
dissolved. If necessary a few more crystals of KI may be added 
to aid solution. Dilute to the mark and mix well. Keep m 
glass-stoppered bottle in cool, dark place. Standardize at once 
against IST/IO sodium thiosulphate solution. Measure out accu- 
rately 25 cubic centimeters of the iodine solution into an Erlen- 
meyer flask, run in sodium thiosulphate until the color is pale 
yellow, then add a few cubic centimeters of a 1 per cent, solu- 
tion of starch (preferably soluble starch) and titrate to disap- 
pearance of blue color. Care should be taken near the end 
point. 

N/25 Tartaric Acid: — ■ 

Tartaric acid 3.0 gm. 

Water to make 1000.0 c.c. 

Equivalent 1 cubic centimeter = 0.0016 gram. 

Preparation- op lSr/20 Permanganate for Potash Solution 
For Folin-Shaffer Uric Acid Test: — 
Dissolve 1.7 grams of potassium permanganate in 1000.0 
cubic centimeters of distilled water and boil for a few minutes 
to increase its permanency. After cooling titrate against a 
N/10 oxalic acid solution (6.3 grams pure oxalic acid crystals 
in 1000.0 cubic centimeters of distilled water.) Pipette exactly 
10.0 cubic centimeters of N/IO oxalic acid into a beaker dilute 
with about 100.0 cubic centimeters of distilled water and add 
15.0 cubic centimeters cone, sulphuric acid. This raises the 
temperature of the solution to about 150 degrees. While hot 
titrate with the permanganate solution under constant stirring 



33 Determination of acetone and acetoacetic acid, p. 533, and determination of 
amylase in feces, p. 248. 



STANDAED SOLUTIONS. 493 

until a uniform red color appears and persists for a few seconds. 
This denotes the end reaction. As the permanganate solution 
was purposely made too strong, less than 20.0 cubic centimeters 
should have been required to produce the end reaction. The 
remaining permanganate solution should be accurately meas- 
ured and then diluted with distilled water until exactly 20.0 
cubic centimeters will give the end reaction with 10.0 cubic 
centimeters of N/10 oxalic acid solution. The permanganate 
solution if well stoppered in a dark bottle kept in a dark place 
should keep for several months, but should from time to time 
be retitrated to insure its accuracy. 

STANDARD SOLUTIONS. 

Standaed Silver Niteate Solution": — 

Silver nitrate . 29.06 gm. 

Distilled water 1000.00 c.c. 

Chemical equivalent: Each 1.0 c.c. equals 0.01 gram 
sodium chloride or 0.006 gram chlorine. 

Potassium Sulphocyanate S/KCNS Standard.^* — ^ 
solution of potassium sulphocyanate is used in the determination 
of the chlorids in the urine and other fluids by the Volhard 
method. A special solution containing 16.6 grams of the sulpho- 
cyanate to the liter, which corresponds to a special silver nitrate 
solution 1 cubic centimeter of which is equivalent to 1 centigram 
of sodium chlorid, is prepared. 

As the potassium sulphocyanatei is hygroscopic, a standard 
solution cannot be made up by direct weighing, but only by 
titration against a previously prepared standard silver solution. 
The reaction between silver nitrate and potassium sulphocyanate 
is AglSrOs + KSCN = AgSCN -f KNO3. In other words, 
one molecule of silver nitrate weighing 169.97 is precipitated by 
a molecule of potassium sulphocyanate weighing 97.22. As the 
silver nitrate solution corresponding to 1 centigram of sodium 
chlorid contains 29.06 grams to the liter, the sulphocyanate 
solution which corresponds to this sliould be 16.6 grams to the 
liter. 



34 For cholesterol in urine, p. 292. 



494 APPENDIX. 

For the special silver nitrate solution S/AgNOs, corre- 
sponding to 1 centigram of sodium chlorid contains 169.97, 
divided by 5.85, which equals 29.06 grams to the liter, so the 
corresponding potassium sulphocyanate solution would contain 
97.32, divided by 5.85, which ejquals 16.6 grams to the liter. 

About 18 grams of the sulphocyanate are weighed out and 
dissolved in about 900 cubic centimeters of water. Ten cubic 
centimeters of the standard silver solution are diluted to 100 
with water. Four cubic centimeters of nitric acid and 5 cubic 
centimeters of ammonioferric alum solution are added and the 
mixture is titrated with the potassium sulphocyanate of un- 
known strength. The end-reaction is marked by the production 
of a slight red color, which remains on stirring the fluid. Inas- 
much as the sulphocyanate solution has been purposely made too 
strong, less than 10 cubic centimeters will be required to 
neutralize 10 cubic centimeters of the silver solution. If we 
assume that 9.8 cubic centimeters of the sulphocyanate are used, 
then it is evident that to each 9.8 cubic centimeters of the solu- 
tion 10 minus 9.8 cubic centimeters, or 0.2 cubic centimeter 
of water, should be added to bring the solution to the proper 
strength. Nine hundred and eighty cubic centimeters of the 
fluid are measured off and filled up to a thousand with distilled 
water. Or, 1000 cubic centimeters are measured off and 24.08 
cubic centimeters of distilled water are added from a burette. 
The contents of the flask is thoroughly shaken; a fresh sample 
is withdrawn and titrated against another 10 cubic centimeters 
of silver nitrate. The second dilution will probably require only 
the addition of a few cubic centimeters of water, in order to 
bring it to the standard. If preferred, however, the solution 
may be left of an arbitrary strength and the correction made at 
the time of titrating the urine. 

SPECIAL REAGENTS AND SOLUTIONS. 

Cream-testing Solutions^-' : — 

"A^^ — Amylic alcohol 37 parts by Tolume. 

Methyl alcohol 13 parts by volume. 

Hydrochloric acid 50 parts by volume. 

"B"— Sulphuric acid. sp. gr. 1832. 

35 Determination of fat in human milk, p. 404. 



SPECIAL REAGENTS AND SOLUTIONS. 495 

Casein Precipitation Solution^^ : — 

Glacial acetic acid 1.0 c.c. 

Alcohol (95 per cent.) 50.0 c.c. 

Distilled water 50.0 c.c. 

Bang's Solution.^'^ — (a) One hundred grams of potassium 
bicarbonate are dissolved in about 1300 cubic centimeters of 
distilled water contained in a 2-liter flask. To this solution are 
added 500 grams of potassium carbonate and 400 grams of 
potassium sulphocyanate. Exactly 25 grams of pure copper 
sulphate (CUSO4, 5H2O) are then dissolved in about 150 cubic 
centimeters of warm distilled water. After cooling, this solution 
is added gradually to the carbonate solution. Add water 
up to the 2-liter mark, allow to stand for twenty-four hours, 
and filter. This solution is stable for about three months. 

(h) Two hundred grams of potassium sulphocyanate are 
dissolved in about 1500 cubic centimeters in a 2-liter flask; 
6.55 grams of hydroxylamin sulphate are dissolved in water and 
added gradually to the sulphocyanate solution. Add water 
up to the 2-liter mark and preserve the mixture in dark-colored 
bottles. This solution is very permanent. 

Benedict's Solutionis : — 

Copper sulphate (C. P. crystallized) 17.3 gm. 

Sodium or potassium citrate 173.0 gm. 

Sodium carbonate (crystallized) or one- 
half the amount of the anhydrous 

salt may be used . . .,. , 200.0 gm. 

Distilled water , q. s. ad 1000.0 c.c 

Dissolve the citrate and carbonate (with aid of heat) in 
about 700 cubic centimeters of water and filter if necessary. 
Dissolve the CUSO4 in about 100 cubic centimeters of water 
and pour into the alkaline solution. Cool and make up to 1 
liter. 



36 Gross's method for tryptic activity, p. 233. 

37 Quantitative determination of glucose, p. 333. 

38 Quantitative determination of glucose, p. 332, 



496 APPENDIX. 

BEXZDicrs Xew Solutiox39 . 

Copi^er sulphate (cryst.) 18.0 gm. 

SodiTun carbonate 200.0 gm. 

Or Sodium carbonate (anhydrous) 100.0 gm. 

Potassium sulphocyanate 125.0 gm. 

Potassium ferrocyanide (5 per cent. soL) . 5.0 c.c. 

Distilled water to make 1000.0 c.c. 

Directions. — Dissolve with the aid of heat the sodium car- 
bonate and sulphocyanate in 800.0 cubic centimeters of water, 
and filter. Dissolve separately the copper sulphate in 100.0 
cubic centimeters of water and pour this mixture slowly into the 
other solution and add the ferrocyanide solution with constant 
stirring and then dilute up to exactly 1000.0 cubic centimeters. 
Equivalent 25 cubic centimeters of the reagent will be reduced 
by 0.050 grams of glucose or by 0.053 grams of levulose. 
Decolorizing Solutiojs's^o . — 

I. Acetic acid, 0,5 to 5.0 per cent, watery solution. 
II. ISTitric acid, 20 to 30 per cent. 
III. Acid alcohol: — 

Sulphuric acid (cone.) 2.0 c.c. 

Alcohol (95 per cent.) 50.0 c.c. 

VTater 150.0 c.c. 

DiAZO Eeagext-^1 : — 

This reagent consists of two solutions which are kept 
separate until mixed for the test. 

Solution ^'^A" — Sulphanilic acid 1.0 gm. 

Hydrochloric acid (con.) . . 50.0 c.c. 

Water 1000.0 c.c. 

Solution "B"' — Sodium nitrate 1.0 c.c. 

Water 200.0 c.c. 

Proportion for test: "A," 5 c.c. '^B/' 3 drops 
Esbach's Eeagext^2 . — 

Picric acid 10.0 gm. 

Citric acid 20.0 gm. 

Water 1000.0 c.c. 

39 Quantitative determinatioii of glucose, p. 332. 
*o Staining for tubercle bacillus, p. 32. 

41 Test for typhoid fever, p. 365. 

42 Quantitative determination of albumin, p. 32-i. 



SPECIAL REAGENTS AND SOLUTIONS. 497 

Feeling's Eeagent^s : — 

The reagent consists of two solutions which are kept 
in separate bottles until mixed immediately before using. 

Solution ^^A" — Copper sulphate 34.64 gm. 

Water 500.00 c.c. 

Solution '^" — Sodium-potassium tartrate . 173.00 gm. 

Sodium hydroxid 125.00 gm. 

Water 500.00 c.c. 

These solutions are used in equal parts for the test. 

FoLiN"- Shaffer Eeagents^^ : — 

A. Ammonium sulphate 500.0 gm. 

Uranium acetate 5.0 gm. 

Distilled water to make 650.0 c.c. 

Dissolve and add 

Acetic acid (10 per cent.) 50.0 c.c. 

Distilled water to make 1000.0 c.c. 

B. Ammonium- sulphate 100.0 gm. 

Distilled water to make 1000.0 c.c. 

C. N/20 potassium permanganate. (See page 492.) 
Equivalent 1 c.c. = 3.75 milligrams uric acid. 

GrUNZBEEG^S PhLOROGLUCIN VaNILLIN^^ : — 

Phloroglucin 2.0 gm. 

Vanillin 1.0 gm. 

Alcohol 30.0 c.c. 

This solution, if active, is pale yellow. It darkens and 
deteriorates with age, especially on exposure to light, so should 
be kept in colored bottles and made fresh from time to time. 

Gross's Casein Solution^^: — 
Reagents. 

{a) Purified casein (G-ruber) 1.0 gm. 

Sodium carbonate 1.0 gm. 

Distilled water 1000.0 c.c. 

(&) Sodium carbonate 1.0 gm. 

Distilled water 1000.0 c.c. 



43 Quantitative determination of glucose, p. 327 

44 Quantitative determination of uric acid in urine, p. 304. 

45 Qualitative determination of free hydrochloric acid, p. 218. 

46 Determination of pancreatic activity, pp. 233 and 247. 

32 



498 APPENDIX. 

Haine's Solution^'^: — 

Copper sulphate 12.0 gm. 

Potassmm hydrate 45.0 gm. 

Grlycerin 90.0 c.c. 

Water q. s. ad 1000.0 c.c. 

A perfectly clear, transparent, dark-blue liquid results 
which throws down a very slight reddish deposit of cuprous oxid 
on standing a week or more. This does not affect the value 
of the solution, as the clear blue solution is simply decanted as 
required. 

Indicators : — 

Congo Eed. — Dissolve 0.5 gi'am of congo red in 90.0 cubic 
centimeters of distilled water and add 10 cubic centimeters of 
95 per cent, alcohol. 

Dl-METHYL-AMIDO-AZOBBNZOL : 

Di-methyl-amido-azobenzol 0.5 gm. 

Alcohol (95 per cent.) 100.0 c.c. 

Methyl Orange: — 

Methyl orange 0.05 gm. 

Distilled water 100.00 c.c. 

Alizarin : — 

Sodium (mono.) alizarin sulphanate .... 1.0 gm. 

Distilled water 100.0 c.c. 

Dissolve the dye in the water and filter from the undis- 
solved residue. 

The stain does not keep well. 

Phenolphthalein : — 

Phenolphthalein 1.0 gm. 

Alcohol (50 per cent.) 100.0 c.c. 

Methyl Eed. — Saturated solution in 50 per cent, alcohol. 

Neutral Red. — A 1 per cent, solution in 50 per cent, 
alcohol. 

47 Qualitative determination of glucose, p. 329 



SPECIAL REAGENTS AND SOLUTIONS. 499 

P-NiTKOPHENOL. — ^A 1 per cent, solution in 50 per cent, 
alcohol. 

Topfer's Eeagent. — Dissolve 0.5 gram di-methyl-amino- 
azo benzene in 100 cubic centimeters 95 per cent, alcohol. 

Teop^olin-00. — ^Dissolve 0.05 gram tropseolin-OO in 100 
cubic centimeters 50 per cent, alcohol. 

Iodine Potassium Iodide Solutionis : — 

Iodine 1 part. 

Potassium iodide 3 parts. 

Gum arabic 50 parts. 

Water 100 parts. 

IVNOP'S SOLUTION'i^ : — 

Bottle "A'^— Sodium hydrate sol 1:25 

Bottle "B"— Bromine 1 part. 

KBr 1 part. 

Water 8 parts. 

For the test add 1 part of the bromine solution to 15 
or 20 parts of the sodium hydrate solution. 

Lugol's Iodin Solution'5^ : — 

lodin 2.0 gm. 

Potassium iodid 4.0 gm. 

Water q. s. ad 100.0 gm. 

Lange's Colloidal Gold Solution^! : — 

Preparation. — Place 500.0 cubic centimeters of double dis- 
tilled water in a 1000-cubic centimeter Jena flask^ and heat 
slowly. When at about 60° 0. add while continuing to heat, 5 
cubic centimeters of 1 per cent solution of gold chloride 
(Mercks C. P.) follow immediately with 5 cubic centimeters of a 
2 per cent, solution of potassium carbonate. Now bring rapidly 
to boiling and remove the flame. If the above technic has been 
carefully followed and if the amounts employed have been accu- 

48 For iodophilia of the blood, p. 85, detection of alcohol in gastric contents, 
p. 225, and Ruhemann's test foiP uric acid, p. 305. 

49 Quantitative estimation of urea in urine, p. 298. 

^0 Detection of starch in gastric contents, p. 225, and detection of acetone 
in urine, p. 344. 

51 For detection of cerebrospinal syphilis, p. 380. 



500 APPENDIX. 

lately measured, then upon vigorously shaking the flask, a serial 
change of color in thei solution will occur, commencing with a 
darkening of the fluid, then passing from faint blue to dark 
blue, purple and finally to red. The solution should be abso- 
lutely clear. Preserve this reagent in a dark glass bottle. 

Lead Acetate (Basic) Solution"^^ . — 

Lead acetate 18.0 gm. 

Litharge 11.0 gm. 

Distilled water to make 100.0 c.c. 

Much's Solution. 53 — Ten cubic centimeters of a saturated 
alcoholic solution of Gruebler^s methyl violet, B. N., mixed with 
90 cubic centimeters of a 2 per cent, aqueous carbolic acid 
solution. 

Magnesia Mixtuee^^ : — 

Ammonium chlorid 10.0 gm. 

Magnesium sulphate 10.0 gm. 

Ammonia water , . 10.0 c.c. 

Water 90.0 c.c. 

The salts are dissolved in the water and the ammonia 
water then added. 

ISToGUCHi's Pyridin Mixture^s . — 

Pyridin 10.0 c.c. 

Formalin 10.0 c.c. 

Acetone 25.0 c.c. 

Alcohol (absol.) 25.0 c.c. 

Water (dist.) 30.0 c.c. 

NoGUCHi's Pyrogallol Solution^^ : — 

F'our per cent. aq. sol. pyrogallol 95.0 c.c. 

Formalin . . ., ., 5.0 c.c. 



52 Quantitative determination of indican, p. 289. 

53 Determination of granular forms of tubercle bacilli, 

54 Detection of earthy phosphates in urine, p. 285. 

55 Detection of Treponema pallidum in tissue, p. 177. 

56 Detection of Treponema pallidum in tissue, p. 177. 



SPECIAL REAGENTS AND SOLUTIONS. 501 

Nylander's Eeagen't^'^ : — 

Bismuth subnitrate 2.0 gm. 

Eochelle salt , . ., 4.0 gm. 

Sodium hydroxid (8 per cent, sol.) 100.0 c.c. 

Xessler-Winkler SoLUTioisr^s : — 

Mercuric iodide i 10.0 gm. 

Potassium iodide . 5.0 gm. 

Sodium hydrate 20.0 gm. 

Distilled water 100.0 c.c. 

Obermeter's Rbagent^^ : — 

Ferric chlorid 3.0 gm. 

Hydrochloric acid 1000.0 c.c. 

Ph. Concentration Solutions^^ • — 

(a) 1/15 mol. potassium di-hydrogen phosphate. Dissolve 
9.078 grams pure crystallized salt in 1000.0 cubic 
centimeters fresh distilled water. 
(h) 1/15 mol. di-sodium hydrogen phosphate. Dissolve 
11.876 grams pure desiccated salt in 1000.0 cubic 
centimeters fresh distilled water. This solution 
should give a strong alkaline reaction to the 
"phthalein indicator.^^ 

Permanganate oe Potash (dilute solution) ^i; — 

Permanganate of potass 3.0 gm. 

Water 1000.0 c.c. 

Keep in colored bottle. 
For use dilute 1 part solution with 39 parts distilled water. 
Approximate value 1 cubic centimeter = 0.15 milligrams indigo. 
Phosphate Chloride Solution^^ . — 

0.2 mol. di-sod. hyd. phos. and 0.1 mol. di- 
hyd. sod. phos. in 1 per cent, sodium 
chloride 1000.0 c.c. 



5" Detection of glucose in urine, p. 330. 

^8 Quantitative determination of non-protein nitrogen, p. 113. and Foliu- 
Farmer method for total nitrogen, p. 313. 

59 Detection of indican in urine, p. 289. 

60 Determination of alkali reserve of the blood, p. 118. 

61 Quantitative determination of indican in the urine, p. 290. 

62 Determination of carbon dioxide tension of alveolar air, p. 12G. and Hawk"? 
method for amylolytic activity of the feces, p. 247. 



502 APPENDIX. 

Pteogallol Solutiox63 : — 

Four per cent. aq. sol. pvrogallol 90.0 c.c. 

Acetone (C. P.) 10.0 c.c. 

PjTidin 1,5.0 c.c. 

Puedy's Eeagext'5-^ : — 

Potassium ferrocyanid 30.0 gm. 

Strong acetic acid 30.0 c.c. 

Water 30.0 c.c. 

Puedy's Eeagext tor Sugae^s • — 

Copper sulphate 4.72 gm. 

Glycerin 38.00 c.c. 

Water 200.00 c.c. 

These should be dissolved in the water by gentle heat. 

Potassium hydroxid 23.50 gm. 

Water 200.00 c.c. 

Dissolve separately and then add to the copper solution. 

When cold add: — 

Ammonia hydroxid (strong) 450.00 c.c. 

Water q. s. 1000.00 c.c. 

EUHEMAXX'S lODIX SOLUTIOX^^ : 

lodin 0.50 gm. 

Potassium iodic! 1.25 gm. 

Absolute alcohol T.50 c.c. 

Glycerin 5.00 c.c. 

Distilled water q. s. ad 100.00 c.c. 

Sahli's Eeagext^" : — 

Potassium iodate (8 per cent, sol.) 50.0 c.c. 

Potassium iodide (48 per cent, sol.) 50.0 c.c. 



63 Detection of Treponema pallidum in tissue, p. 177. 

64 Determination of albumin in the urine, p. 323. 

65 Quantitative determination of glucose in urine, p. 333. 

66 Quantitative determination of uric acid, p. 305. 

6T Quantitative determination of free hydrochloric acid, p. 205. 



SPECIAL REAGENTS AND SOLUTIONS. 503 

Stokers Solutioj^^^ : — 

Ferrous sulphate 2.0 gm. 

Tartaric acid 3.0 gm. 

Water 100.0 c.c. 

Staech Solution, One Per Cent.^^ : — 

One gram starch powder, dissolved in cold water and 
stirred to form uniform suspension. Heat slowly and stir until 
clear, when cold add additional water to make 100.0 cubic 
centimeters. 

Silver-pyridin Solution'^ ^ : — ■ 

One per cent. aq. sol. AgN'Og . . .*. 90.0 c.c. 

Pyridin 10.0 c.c. 

Standard Picramic Acid^^: — 

Picramic acid 0.064 gm. 

Sodium carbonate (anhyd.) 0.100 gm. 

Distilled water to make 1000.000 c.c. 

Dissolve picramic acid by aid of heat in 50 cubic centim- 
eters of the water which has been alkalinized by the sodium 
carbonate, when cool add the remainder of the water. 

Tsuchiya's Eea.gbnt'^^ . — 

Phosphotungstic acid 1.5 gm. 

Concentrated HCl 5.0 c.c. 

95 per cent, alcohol . .q. s. ad 100.0 c.c. 

Trommer-Simrock Eeagbnt'^3 : — 

Copper sulphate 2.0 gm. 

Potassium hydroxid (5 per cent, sol.) .... 150.0 c.c. 

Glycerin 15.0 c.c. 

Distilled water 15.0 c.c. 

Tanret's Eeageint'^4 . 

Dissolve 33.1 grams potassium iodide in 200 cubic cen- 
timeters of water. Add 13.5 grams powdered mercuric 

68 Spectroscopic reducing solution, p. 91. 

69 Quantitative determination of amylolytic activity of feces, p. 248. 
'i'O Detection of Treponema pallidum in tissue, p. 177. 

'1 Sims and Benedict's method for blood sugar, p. 131. 
"2 Quantitative determination of albumin in urine, p. 325. 
'3 Qualitative determination of glucose in urine, p. 330. 
"4 Qualitative determination of glucose in urine, p. 323. 



504 APPENDIX. 

cMorid and water, stirring until the red precipitate first 
formed has been dissolved. 

Dilute to 900 cubic centimeters with water and add 
100 cubic centimeters strong acetic acid. Allow to stand 
twelve hours, and then decant from precipitate and use 
clear solution. This forms a solution of mercuric-potassium 
iodid in dilute acetic acid. 

Uffleman^s Reagent'^ ^ : — 

Carbolic acid (4 per cent.) 10 c.c. 

Water 20 c.c. 

Liquor ferri chloridi 1 drop. 

This solution should be a clear amethyst color, and should 
be prepared fresh for use. 

Folin-Farmek Nitrogen Standard Solution. ^ 6 — The 
standard solution used in the colorimetric determination of total 
nitrogen is prepared by dissolving 4.714 grams of dry purified 
ammonium sulphate in 1000 c.c. of ammonia free water. This 
solution is carefully prepared from absolutely pure dry ammon- 
ium sulphate and should have the following value — one cubic 
centimeter equals one milligram of nitrogen. 

NoGUCHi Silver Pyridin Solution''' ^ : — > 

Silver nitrate (1 per cent, aqueous solution) 90.0 c.c. 
Pyridin 10.0 c.c. 

Baybee and Lorenz^s Special Diluting Fluid'^^ . — 

Methyl violet 0.1 gm. 

Glacial acetic acid 2.0 c.c. 

Aq. dest ■ q. s. ad 50.0 c.c. 

Peptone Solution^^ : — 

Prepared by dissolving 1.0 gm. of Witters peptone in 100 
c.c. of distilled water. 

75 Detection of lactic acid in gastric contents, p. 223. 

76 Estimation of total nitrogen, p. 813. 

77 Staining Treponema pallidum in tissue, p. 177. 

78 Baybee and Lorenz's method, p, 382. 

79 Oliver's test for bile aPiJJs, p. 349. 



TABLES. 



505 



TABLES. 



Meihic Weights and Measures.so 



Weights. 

1 millgram 

1 centigram 

1 decigram 

1 gram 

1 decagram 

1 hectogram 

1 kilogram 
Measures. 

1 millimeter 

1 centimeter 

1 decimeter 

1 meter 

1 decameter 

1 hectometer 

1 kilometer _-, . 

1 yard or 36 inches 

1 inch 



0.001 


grams 


0.015 grains Troy. 


01 


** 


0.154 


0.1 




1.543 " 
15.432 " 


10 


*• 


154.324 " 


100 


•* 


0.268 pounds " 


1000 


" 


2.679 •• 


0.001 meter 


0.0394 inch. 


0.01 


•* 


0,3937 " 


0.1 


" 


3.9371 inches. 
39.3708 " 


10 


•• 


32.8089 feet. 


1(10 


" 


328.089 " 


1000 
ches 




0.6214 mile. 
0.9144 meter. 
25.4 millimeters. 



Inteenational Atomic Weights, 1916. 



0=16. 

Aluminum Al 27.1 

Antimony Sb 120.2 

Arsenic As 74.96 

Barium Ba 137.37 

Bismuth Bi 208.0 

Boron B 11.0 

Bromine Br 79.92 

Cadmium Cd 112.40 

Calcium Ca 40.07 

Carbon C 12.005 

Chlorine 01 35.46 

Chromium Cr 52.0 

Cobalt Co 58.97 

Copper Cu 63.57 

Fluorine F 19.0 

Glucinum Gl 9J. 

Gold Au 197.2 

Hydrogen H 1.008 

Iodine I 126.92 

Iridium Ir 193.1 

Iron Fe 55.84 

Lanthanum La 139.0 

Lead Pb 207.20 

Lithium Li 6.94 

Magnesium Mg 24.32 



0=16. 

Manganese Mn 54.93 

Mercury Hg 200.6 

Molybdenum Mo 96.0 

Nickel Ni 58.68 

Nitrogen N 14.01 

Osmium Os 190.9 

Oxygen O 16.00 

Palladium Pd 106.7 

Phosphorus P 31.04 

Platinum Pt 195.2 

Potassium K 39.10 

Radium Pa 226.0 

Selenium Se 79.2 

Silicon Si 28.3 

Silver Ag 107.88 

Sodium Na 23.00 

Strontium Sr 87.63 

Sulphur S 32.06 

Tantalum Ta 181.5 

Tellurium Te 127.5 

Tin Sn 118.7 

Titanium Ti 48.1 

Tungsten W 184.0 

Uranium U 238.2 

Zinc Zn 65.37 



80 From Gould's Pocket Dictionary. 



506 



APPENDIX. 



Multiples of a Geain. 
From 1 grain to 1 ounce 



U. S. A. Metric 

1 0.065 gm. 

11 - 0.086 gm. 

1^ 0.097 gm. 

If 0.113 gm. 

2 0.13 gm. 

2J 0.162 gm. 

3' 0.194 gm. 



gr- 
gr. 
gr- 
gr. 
gr- 
gr. 
gr- 
gr. 3} 0.227 gm. 

gr 
gr 
gr 
gr 
gr 
gr 
gr 



4 0.259 gm. 

5 0.324 gm. 

6 0.389 gm. 

7 0.454 gm. 

8 0.518 gm. 

8| 0.567 gm. 

9 0.583 gm. 

gr. 10 0.648 gm. 

gr. 12 0.778 gm. 



U. S. A. 



Metric 



gr- 
gr. 
gr. 
gr. 
gr. 
gr. 
gr. 
gr. 
gr. 
gr. 
oz. 
oz. 
oz. 
dr. 
oz. 
dr. 



15 0.972 gm. 

18 1.166 gm. 

20 1.296 gm. 

25 1.620 gm. 

30 . 1.944 gm. 

35 2.268 gm. 

40 

50 

60 

120 



2.592 


gm. 


3.24 


gm 


3.89 


gm. 


7.78 


gm 


3.54 


gm 


7.08 


gm 


14.17 


gm 


15.55 


gm 


28.35 


gm 


31.1 


gm 



Equivalents of U. S. A and Metric Measures of Capacity. 
From half-a-minim to 1 fluid ounce 



U. S. A. Metric 

min. i 0.03 c.c. 

min. 1 0.062 c.c. 

min. 2 0.123 c.c. 

min. 3 0.185 c.c. 

min. 4 0.246 c.c. 

min. 5 0.308 c.c. 

min. 6 0.370 c.c. 

min. 7 0.431 c.c. 

min. 8 0.493 c.c. 

rain. 9 0.554 c.c. 

min. 10 0.616 c.c. 

min. 12 739 c.c. 

rain. 15 924 c.c. 



U.S.A. Metric 

min. 20 1.232 c.c. 

min. 25 1.54 c.c. 

min. 30 1.848 c.c. 

min. 35 2.156 c.c. 

min. 40 2.464 c.c. 

min. 50 3.08 c.c. 

min. 60 3.70 c.c. 

min. 90 5.54 c.c. 

min. 120 7.39 c.c. 

min. 180 11.09 c.c. 

min. 240 14.79 c.c. 

min. 360 22.18 c.c. 

min. 480 29.57 c.c. 



In Continental prescribing, a smaller quantity than half a cubic centimeter is 
usually expressed in drops, which, in dispensing, are dropped from pipette into the 
cubic centimeter measure. 



TABLES. 



507 



AppRoxi]\rATE U. S. A. Equivalents of Metric Measure of Capacity. 



Metric 


U. S. A. 


Metric 










U.S. A 


Ic.c... 


16 (16.23) min. 


25 c.c. 






6fl 


dr. 


46 min 


2c.c... 


32j min. 

48f min. 


30 c.c. 
40 c.c. 






. 8fl 
, 2fl, 


dr. 
dr. 


7 mm 


3 c.c... 


. Ifl. 


oz. 


49 min 


4c. c. .. 


1 fl. dr. 5 min. 


50 c.c. 


. Ifl. 


oz. 


, 5fl. 


dr. 


32 min 


5 c.c... 


1 fl. dr. 21 min. 


75 c.c. 


. 2fl. 


oz. 


, 4fl. 


dr., 


17 min 


6 c.c. .. 


Ifl. dr. 37 n.in. 


103 c.c. 


. 3fl. 


oz. 


, 3fl. 


dr., 


3 min. 


7 c.c... 


Ifl. dr. 54 min. 


125 c.c. 


. 4fl. 


oz. 


Ifl. 


dr. 


49 min. 


8 c.c... 


2fl. dr. 10 min. 


150 c.c. 


. 5fl. 


oz. 


Ofl. 


dr., 


35 min. 


9c.c. .. 


2fl. dr. 26 min. 


200 c.c. 


. 6fl. 


oz. 


6fl. 


dr., 


6 min 


10 c.c... 


2fl. dr. 42 min. 


300 c.c. 


.10 fl. 


oz. 


Ifl. 


dr., 


9 min 


12 c.c... 


3fl. dr. 23 min. 


500 c.c. 


.16 fl. 


oz. 


7fl. 


dr., 


15 min. 


15 c.c... 


4 fl. dr. 4 min. 


1 litre.. 


.33 fl. 


oz. 


, 6fl. 


dr. 


31 min 


20c.c. .. 


5 fl. dr. 25 min. 















Approximate U. S. A. Equivalents of Metric Measures of Mass. 



Metric 



U. S. A 



mgm ^'^ gr. 

mgm gL gr. 

mgm 

mgni tV gr 



mgm 

6.5 mgm 

8 mgm 

cgm 

cgm 

cgm 

cgm 

6.5 cgm 

10 cgm 

15 cgm 2} gr. 

20 cgm 3 gr. 

26 cgm 4 gr. 

30 cgm 4\ gr. 

40 cgm 6} gr. 

50 cgm 7f gr. 

75 cgm 11^ gr. 



^V 


gr 


tV 


gr 


tV 


gr 


I'o 


gr 


i 


gr. 


1 


gr. 


i 


gr 


i 


gr. 


1 


gr. 


1 


gr. 


u 


gr. 



Metric 



U.S.A. 



1 gm 151(15.432) gr. 

2 gm 30i gr. 

3 gm 461 gr. 

4 gm 61f gr. 

5 gm 77} gr. 

7.5 gm 115f gr. 

10 gm 154^ gr. 

15 gm 231Jgr. 

20 gm 308|gr. 

25 gm 385igr. 

30 gm 1 oz. 25^- gr. 

40 gm 1 oz. 179i gr. 

50 gm 1 oz. 334 gr. 

75 gm 2oz. 282igr. 

100 gm 3oz. 2301 gr. 

150 gm 5oz. 127igr. 

250 gm 8oz. 358 gr. 

500 gm 1 lb. loz. 278 gr. 

750 gm 1 lb. 10 oz. 200 gr. 

1 kgm 21b. 3 oz. 120 gr. 



508 



APPENDIX. 



Comparison of Thebmometeks.si 



Fahr. 


Cent. 


Reau. 


Fahr. 


Cent. 


Reau. 


212 


100 


80 


76 


24.4 


19.6 


210 


98.9 


79 1 


74 


23.3 


18.7 


208 


97 8 


78.2 


72 


22.2 


17.8 


206 


96 7 


77.3 


70 


21.1 


16 9 


204 


95.6 


76.4 


68 


20 


15 


202 


94.4 


75.6 


66 


18.9 


15.1 


200 


93 3 


74.7 


64 


17.8 


14.2 


198 


92.2 


73.8 


62 


16.7 


13.3 


196 


91.1 


72.9 


60 


15.6 


12.4 


194 


90 


72 


58 


14 4 


11.6 


192 


88.9 


71.1 


56 


13.3 


10.7 


190 


87.8 


70.2 


64 


12.2 


9.8 


188 


86.7 


69.3 


52 


11.1 


8.9 


186 


85.6 


68.4 


50 


10 


8 


184 


84.4 


67.6 


48 


8.9 


7.1 


182 


83.3 


66.7 


46 


7.8 


6.2 


180 


82.2 


65.8 


44 


6.7 


5.3 


178 


81.1 


64.9 


42 


6.6 


4.4 


176 


80 


64 


40 


4.4 


36 


174 


78.9 


63.1 


38 


3.3 


2.7 


172 


77.8 


62.2 


36 


2.2 


1.8 


170 


76.7 


61.3 


34 


1.1 


0.9 


168 


75 6 


60.4 


32 








166 


74.4 


59.6 


30 


-1.1 


-0.0 


16* 


73.3 


58 7 


28 


-2.2 


-1.8 


162 


72.2 


57.8 


26 


-3.3 


-2.7 


160 


71.1 


56.9 


24 


-4.4 


-3.6 


158 


70 


56 


22 


-5.6 


-4.4 


156 


68.9 


55.1 


20 


-6.7 


-5.3 


154 


67.8 


64.2 


18 


-7.8 


-6.2 


152 


66 7 


63.3 


16 


-8.9 


-7.1 


150 


65.6 


62.4 


14 


-10 


-8 


148 


64.4 


516 


12 


-11 1 


8.9 


146 


63.3 


50.7 


10 


-12.2 


-9.8 


144 


62.2 


49.8 


8 


-13.3 


-10.7 


142 


61.1 


48.9 


6 


-14.4 


-11.6 


140 


60 


48 


4 


-15 6 


-12.4 


138 


58.9 


47.1 


2 


-16.7 


-13.3 


136 


67 8 


46.2 





-17.8 


-14.2 


134 


56.7 


45.3 


-2 


-18.9 


-15.1 


1-2 


55 6 


44 4 


-4 


-20 


-16 


130 


54.4 


43.6 


-6 


-21.1 


-16.9 


128 


53.3 


42 7 


-3 


-22.2 


-17.8 


126 


52.2 


41.8 


-10 


-23.3 


-18.7 


124 


51.1 


40.9 


-12 


-24.4 


-19.6 


122 


50 


40 


-14 


-25.6 


-20.4 


120 


48.9 


39.1 


-16 


-26.7 


-21.3 


118 


47.8 


38.2 


-18 


-27.8 


-22.2 


116 


46.7 


37.3 


-20 


-28.9 


-23.1 


114 


45.6 


36.4 


-22 


-30 


-24 


112 


44.4 


35.6 


-24 


-31.1 


-24.9 


110 


4U 


34.7 


-26 


-32.2 


-25.8 


108 


42.2 


33.8 


-28 


-33.3 


-26.7 


106 


41.1 


32 9 


-.30 


-34.4 


-27.6 


194 


40 


32 


-32 


-36.6 


-28.4 


102 


38.9 


31.1 


-U 


-36.7 


-29.3 


100 


37 8 


30.2 


-36 


-37.8 


-30.2 


98 


36.7 


29.3 


-38 


-38.9 


-31.1 


% 


35.6 


28 4 


-40 


-40 


-32 


94 


34.4 


276 


-42 


-41.1 


-32.9 


92 


33.3 


26.7 


-44 


-42.2 


-33.8 


90 


32.2 


25.8 


-46 


-43.3 


-34.7 


88 


31.1 


24.9 


-48 


-44.4 


-35.6 


86 


30 


24 


-50 


-45.6 


-36.4 


84 


28.9 


23.1 


-52 


-46.7 


-37.3 


82 


27.8 


22 2 


-54 


-47 8 


-38.2 


80 


26.7 


213 


-56 


-48 9 


-39.1 


78 


25.6 


20.4 









81 Frcm Gould's New Medical Dictionary. 



Bang's Table of Reduction Equivalents. 
(Webster : ** Diagnostic Methods," 1912.) 



Cubic centimeters of 


Milligrams 


hydroxylamin solution. 


of sugar. 


0.75 


60.0 


1.00 


59.4 


1.50 


58.4 


2.00 


57.3 


2.50 


56.2 


3.00 


55.0 


3.50 


54.3 


4.00 


53.4 


4.50 


52.6 


5.00 


51.6 


5.50 


50.7 


6.00 


49.8 


6.50 


48.9 


7.00 


48.0 


7.50 


47.2 


8.00 


46.3 


8.50 


45.5 


9.00 


44.7 


9.50 


44.0 


10.00 


43.3 


10.50 


42.5 


11.00 


41.8 


11.50 


41.1 


12.00 


40.4 


12.50 


39.7 


13.00 


39.0 


13.50 


38.3 


14.00 


37.7 


14.50 


37.1 


15.00 


36.4 


15.50 


35.8 


16.00 


35.1 


16.50 


34.5 


17.00 


33.9 


17.50 


33.3 


18.00 


32.6 


18.50 


32.0 


19.00 


31.4 


19.50 


30.8 


20.00 


30.2 


20.50 


29.6 


21.00 


29.0 


21.50 


28.3 


22.00 


27.7 


22.50 


27.1 


23.00 


26.5 


23.50 


25.8 


24.00 


25.2 


24.50 


24.6 


25.00 


24.1 



Cubic centimeters of 


Milligrams 


hydroxylamin solution. 


of sugar. 


25.50 


23.5 


26.00 


22.9 


26.50 


22.3 


27.00 


21.8 


27.50 


21.2 


28.00 


20.7 


28.50 


20.1 


29.00 


19.6 


29.50 


19.1 


30.00 


18.6 


30.50 


18.0 


31.00 


17.5 


31.50 


17.0 


32.00 


16.5 


32.50 


15.9 


33.00 


15.4 


33.50 


14.9 


34.00 


14.4 


34.50 


13.9 


35.00 


13.4 


35.50 


12.9 


36.00 


12.4 


36.50 


11.9 


37.00 


11.4 


37.50 ^ 


10.9 


38.00 


10.4 


38.50 


9.9 


39.00 


9.4 


39.50 


9.0 


40.00 


8.5 


40.50 


8.1 


41.00 


7.6 


41.50 


7.2 


42.00 


6.7 


42.50 


6.3 


43.00 


5.8 


43.50 


5.4 


44.00 


4.9 


44.50 


4.5 


45.00 


4.1 


45.50 


3.7 


46.00 


3.3 


46.50 


2.9 


47.00 


2.5 


47.50 


2.1 


48.00 


1.7 


48.50 


1.3 


49.00 


0.9 


49.50 


0.5 


50.00 


0.0 



For every 0.1 c.c. hydroxylamin solution used more than is 
given in the table, subtract 0.1 mg. if the reading be between 49 
and 15, while if it be between 15 and 1 subtract 0.2 mg. 

! (509) 



510 



APPENDIX. 



Blood Peessube Chaet. 

A new graphic chart for recording blood-pressure observa- 
tions. This chart will be found very valuable in connection with 
serial studies of blood-pressure where it is desired to follow 



PULSE, TEMPERATURE AND BLOOD PRESSURE CHART 

CHART HO.^^ If Dnitned by FrucU A. Fiotbt. M. D. 






COLOR. ^■" 



"" ^ ^ 


t.. 








•^ ^;:i,'-.v.';^t.i2" ~- :"s.c..H*^ rix*.-^. 


DIAGNOSIS (P/v_t.v^^^-~0-v>*. 




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135 135 










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65 65 


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PULSE PRESSURE 


iX 


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FIG. 78.— Specimen of a New Graphic Chart for Recording Blood 
Pressure Observations, Arranged for Daily, Weekly or Irregular 
Observation Periods. Spaces Equal 5 Millimeters. 

from time to time its fluctuations. It is constructed along the 
lines of a temperature chart. The changes in blood-pressure 
can be readily followed, and the effect of treatment more cer- 
tainly demonstrated. 



TABLES. 



511 



Table for Converting Apothecaries' Weights and Measures 
INTO Grams. 82 



Troy 
Weight. 


Metric. 




Grams for Liquids. 






Apothecaries" 












Measure. 


Lighter 


Specific 


Heavier 


Grains. 


Grams. 




tiian 


Gravity 


than 








Water. 


of Water. 


Water. 


1-400 


.00016 


m 1 


.055 


.06 


.08 


1-200 


.00033 


2 


.10 


.12 


.15 


1-12S 


.0005 


3 


.16 


.18 


.24 


1-100 


.000C5 


4 


.22 


.24 


.32 


1-64 


.001 


5 


.28 


.30 


.40 


1-40 


.0015 


6 


.32 


.36 


.48 


1-30 


.002 


7 


.38 


.42 


.55 


1-20 


.003 


8 


.45 


.50 


.65 


1-16 


.004 


9 


.50 


.55 


.73 


1-12 


.005 


10 


.55 


.60 


.80 


1-10 


.0U6 


15 


.80 


.72 


.96 


1-8 


.008 


16 


.90 


1.00 


1.32 


1-3 


.010 


20 


1.12 


1.25 


1.60 


1-4 


.016 


2> 


1.40 


1.55 


2.00 


1-4 


.02 


30 


1.70 


1.90 


2.50 


1-4 


.03 


35 


2.00 


2.20 


2.90 


1 


.065 


40 


2.25 


2.50 


3.30 


2 


.13 


48 


2.70 


3.00 


400 


3 


.20 


50 


2.80 


3.12 


4.15 


4 


.26 


60f5j 


3.40 


3.75 


5.00 


5 


.32 


72 


4.00 


4.50 


6.00 


6 


.39 


80 


4.50 


5.00 


6.65 


8 


.52 


90 


5.10 


5.60 


7.50 


10 


.65 


96 


5.40 


6.00 


8.00 


15 


1.00 


100 


5.60 


6.25 


8.30 


20 9 j 


1.30 


120 fSij 


6 75 


7.50 


10.00 


24 


1.50 


160 


9.00 


10.00 


13.30 


26 


1.62 


180 f5ii,j 


10.10 


11.25 


15.00 


30 5SS 


1.95 


240 f5s3 


13..50 


15.00 


20.00 


40 


2 60 


f5v 


16.90 


18.75 


25.«0 


50 


3.20 


f5vj 


20.25 


22.50 


30.00 


60 5.i 


3.90 


f5vij 


23.60 


26.25 


35.00 


120 5ij 


7 80 


fsj 


27.00 


30.00 


40.00 


180 


11.65 


fsii 


54.00 


60.00 


80.00 


240 5s8 


15.50 


fSiij 


81.0) 


90.00 


120.00 


300 


19.40 


f5iv 


108.00 


120.00 


160.00 


360 


23.30 


f5v 


1X5.00 


150.00 


i 00.00 


42j 


57.20 


fSvj 


162.00 


180.00 


240 00 


480 Sj 


31.10 


fSviij 


216.00 


240.00 


320.00 



82 From Gould's Pocket Dictionary. 



INDEX. 



Absorption rate determination of gas- 
tric mucosa, 235 

Penzoldt's method, 235 
Absorption spectrum, 90 
Acetic acid in stomach, 224 

test for exudates, 394 

test for albumin, 321 
Acetic-guaiac test for occult blood, 

227 
Aceto-acetic acid in urine, 344 
Acetone in urine, 343 

ethylene diamin hydrate test for, 343 

Frommer's test for, 343 

Gumming's test for, 344 

Legal 's test for, 343 • 

Lieben's test for, 344 
Achorion Schonleinii, 197 
Actinomycosis, pulmonary, 30 
Agar-agar, 419 
Agglutination reactions, 438 

cholera, 442 

colon bacillus, 442 

dysentery, 442 

malta fever, 443 

paratyphoid, 441 

plague, 442 

Widal's,- 438 
Albuminuria, 319 

extrarenal, 321 

transient, 319 
Albumin content of sputura, 43 
Albumin, estimation of, in cerebro- 
spinal fluid, 385 

Lange's colloidal gold test for, 386 

Nonne's test for, 385 
Albumin in urine, 319 

qualitative tests for, 321 

quantitative tests for, 323 
Alcohol in stomach contents, 225 
Alkaline hematin, 91 
Alkaline phosphates in urine, 286 
Alkaline reserve of blood plasma, 118 

sign;ificance of, 123 
Alkaline urine, 282 
Alveolar carbon dioxide tension, 124 

collection of sample, 124, 128 

Marriott's method, 125 
Ameba histolytica, 180 

description of, 180 

examination for, 180 
Amebic dysentery, 264 
Ammoniacal urine, 273 
Ammonia in urine, 314 

Folin's method for, 316 

formalin test for, 315 

Schlosing's test for, 314 
significance of, 314 
Ammonium urate in urine, 363 
Amplitude of blood-pressure, 134 
Amount, normal, of urine, 273 
Analysis of concretions, 364 
Anemia, primary, 79 
Aneroid sphygmomanometer, 140 



Animal parasites, 177 

bothriocephaloidia, 186 

classification of, 178 

flagellata, 181 

infusoria, 184 

nematodes, 189 

platyhelminthes, 184 

protozoa, 179 

rhizopodia, 180 

sporozoa, 183 

temporary, 177 

tenidae, 186 
Ankylostoma duodenale, 192 
Antiformin method, 35 
Antigen, preparation of, 454 

luetic, 447 

titration of, 455 
Antihuman amboceptor, 456 
Anti-sheep amboceptor, 452 
Anuria, 275 

Apochromatic objective, 5 
Arachnoidea, 194 
Arneth's classification of leukocytes, 

75 
Arnold's steam sterilizer, 414 
Aiterial pressure, 134 
Arthropodia, 194 
Ascaris lumbricoides, 193 
Ascitic fluid, varieties of, 398 
Auscultatory blood-pressure reading, 

143 
Autenreith and Funk's method for 

cholesterol, 116 
Autoclave sterilization, 412 



Bacillus coli communis in feces, 261 

diphtheria, 428 

influenza, 40 

tuberculosis, 261 

typhosis, 430 
blood cultures of, in feces, 131 
Bacteria, classification of, 432 

in feces, 260 
Bacteriologic methods, 407 

culture-tube filling, 422 

differential methods, 432 

incubation, 424 

plate-making, 423 

preparation of culture media, 416 

separating organisms, 426 

sterilization of media, 407 
Bacterial casts, 356 
Bacterinuria, 360 
Balsams in urine, 369 
Bang's test for glucose, 333 
Bence-Jones albumin, 325 
Benedict's quantitative test, 332 
Benzidin test for occult blood, 228 

test for blood in stool. 256 
Benzaldehyd reaction of Ehrlich. o77 
Bile in the blood, 85 
tests for, 85 

in gastric contents, 235 



(.513) 



514 



INDEX. 



Bile in the urine, 346 
Bile acids, tests for, 348 

Oliver's, 349 

Pettenkofer's, 349 
Bile-pigments, tests for, 346 

in gastric juice, 236 

Gmelin's, 346 

methylene blue, 347 

Nakayama's, 348 

nitric acid, 347 

Rosenbach's, 348 

Smith's, 347 
Bilharzia, 185 
Bilirubin in, feces, 260 
Bird's formula for total solids, 279 
Blood, the, 45 

amount of, 48 

anemias of, 78 

appearance of, 46 

bile in, 85 

chemical composition of, 49 

cholesterol content of, 114 

chlorosis, 80 

coagulation time of, 95 
Bogg's method, 98 
Dorrance's, 98 
Milan's, 97 
Russel and Brodie's, 97 

color of, 46 

color index of, 61 

counting diaphragm for, 64 

degeneration forms of, 83 

differential count of, 72 

examination of, 50 

examination of corpuscles of, 56 

hemoglobin estimation of, 51 

iodophllia in, 85 

leukemia, 81 

leukocyte count of, 63 

leukocytes in, 71 

leukocytosis, 77 

making slides of, 66 

microscopic examination of, 65 

non-protein nitrogen of, 112 

odor of, 47 

percentage of cells in, 61 

pseudoleukemia, 83 

reaction of, 48 

red-cell count of, 59 

specific gravity of, 47 

staining smears of, 68 

taste of, 47 

total nitrogen of, 113 

tubercle bacilli in, 427 

viscosity of, 102 

vital staining of, 84 

volume index of, 62 
Blood-casts, 356 
Blood-cells in urine, 359 
Blood-cultures, 431 
Blood examination, microscopic, 15 
Blood in feces, tests for, 254 

Benzidin, 256 

de Jager's, 257 

guaiac, 255 

Kliinge's, 256 

phenolphthalein, 258 

Van Deen's, 255 
Blood plasma, 118 

alkaline reserve of, 118 

hydrogen-ion tension of, 121 
Levy, Rowntree and Marriott's 
method for, 121 
Blood platelet count, 64 
Blood-pressure, the, alcohol on, 150 

auscultatory method for, 143 



Blood-pressure, diastolic, 134, 142 

diet on, 150 

exercise on, 149 

emotions on, 149 

factors influencing, 146 

hypertension, 151 

hypotension, 151 

in life insurance examination, 153 

mean pressure, 135 

myocarditis, effect on, 151 

palpating method for, 142 

pathologic, 151 

posture on, 148 

pulse pressure, 134, 145 

recording of, 146 

systolic, 134 

tobacco, effect on, 150 
Blood serum media, 420 
Blood smear preparations, 66 

staining of, 68 
Blood stains, 68 

Ehrlich's method, 68 

eosin-hematoxylin, 70 

Romanowski, 68 

universal, 68 
Blood in stools, 244 

significance of, 259 
Blood in urine, 350 

appearance of, 351 

test for, 351 
Blood sugar, 129 

Epstein's method for, 129 

Lewis and Benedict's method, 131 
Pearce's modification of, 132 
Blood urea, 107 

method of Marshall, 107 

method of Rose and Coleman, 111 

method of Van Slyke and Cullen, 
110 
Boas-Oppler bacillus in stomach, 226 
Boas's test for free hydrochloric acid, 

219 
Boas's test for occult blood, 227 
Boas's test for motor function, 237 
Boas's test-meal, 210 
Body fluids, staining bacteria in, 436 
Boggs's coagulometer, 98 
Boggs's method for milk protein, 405 
Bordet-Gengou bacillus, 42 
Bottger's bismuth test, 330 
Bothriocephaloidia, 186 
Bouillon culture media, 416 
Bromin iui urine, 368 
Bronchitis, 25 
Butyric acid in stomach contents, 224 



Calculi in feces, 244, 265 

Camera lucida, 16 

Cammidge reaction, 376 

Capacity of stomach, 215 

Capillary blood-pressure, 134 

Capsule staining, 40, 436 

Carbohydrate conversion power of 

gastric juice, 235 
Carbonates in urine, 363 
Carbon dioxid hemoglobin, 90 
Carbon dioxid tension of alveolar air, 

124 
Care of microscope, 9 
Casts in urine, 353 

in sputum, 21 

varieties of, 355 
Cell count, spinal fluid, 381 

blood, 56 



INDEX. 



515 



Cerebrospinal fluid, 378 
albumin determination of, 385 
chemical examination of, 379 
collection of specimen of, 381 
cytology of, 381 
differential count of, 383 
physical characteristics of, 379 
pressure reading of, 380 
protein determination of, 384 
tubercle bacillus In, 387 
Cercomonas Intestinalis, 182 
Charcot-Leyden crystals in sputum, 

28 
Chart for blood count, 73 
Chemical composition of the blood, 43 
Chemical composition of urine, 282 
Chemical examinations, of the blood, 
384 
of human milk, 404 
of the feces, 249 
of the urine, 283 
Chemical sterilization, 415 
Chemistry of digestion, 217 
Cholesterin in urine, 363 
Cholesterol content of the blood, 114 
Autenreith and Funk's method, 116 
Meyers's method, 114 
Chlorids in urine, 292 
approximate test, 292 
Purdy's test, 292 
Salkowski-Volkard test, 292 
Chlorophil, 90 
Chlorosis, 80 

Cholera, agglutination test for, 442 
Cholera bacillus in feces, 263 
Chylous exudates, 395 
Chyluria, 360 
Cimex lectularius, 196 
Classification, of bacteria, 432 
Gram's method, 432 
Wright's method, 432 
Clinical hematology terms, 70 
Coagulation time of blood, 95 
Coal-tar products in urine, 368 
Coccidium hominis, 183 
Collecting blood specimens, 112 
milk specimens, 402 
urine specimens, 268 
Colon bacillus agglutination, 441 
Colorimeter, 371 
Color index, 61 
Color of spiutum, 21 
Color of urine, 271 
Complement, preparation of, 454 
Composition of mother's milk, 405 
Concretions in feces, 265 
in sputum, 25 
in urine, 364 
Conjunctival secretions, 391 
gonococcus in, 391 
Koch-Weeks bacillus in, 391 
trachoma shown by, 391 
Councilman and Mallory media, 421 
Counting diaphragm for blood cells, 

64 
Creatinin in urine, 309 
Jaffe's test for, 309 
Weyl's test for, 309 
Crystalline casts, 357 
appearance of, 357 
examination for, 358 
Culture media, preparation of. 416 
agar- agar, 419 
blood serum, 420 
bouillon, 416 
gelatin, 418 



Culture media, glycerin-agar, 419 

litmus agar, 420 
Culture-tube filling, 422 
Curschmann's spirals, 27 
Cyanhemoglobiui, 90 
Cylindroids, in urine, 357 
Cyst fluids, 398 

echinococcus, 398 

ovarian, 398 

pancreatic, 399 
Cysticercus acanthotrias, 187 
Cystin in urine, 317, 363 
Cystology of exudates, 397 
Czaplowsky's staining method, 33 



Daland-Faught test meal apparatus, 

211 
de Jager's test for blood in stool, 257 

for glucose, 329 
Dare's method for hemoglobin, 55 
Dark-ground illuminaUonj, 7 
Decomposition changes in urine, 269 
Degeneration forms of blood, 83 
Demodex folliculorum, 194 
Denning and Watson's method, 106 
Detection of lead in urine, 367 

antipyrin in urine, 368 

balsams in urine, 369 

bromin la urine, 368 

iodin in urine, 367 

mercury in urine, 367 

phenol in urine, 368 

santonin in urine, 369 

tannin in urine, 369 
Determination of sugar in milk, 405 

protein in milk, 405 

fat in milk, 404 
Diacetic acid in urine, 345 

Gerhardt's test for, 345 
Diastolic blood-pressure, 134, 142 
Dialyzing sacs for H-ion concentra- 
tion, 121 
Diazo reaction, 365 
Differential blood count, 72 

spinal fluid count, 383 
Jjifferenjtial density method for glu- 
cose, 336 
Differentiation of white cells, 69 
Diphtheria bacillus, 389, 428 
Diphtheria, specific reaction, 464 
Disinfection, 407 
Distomum hematobium, 185 
Distomum hepaticum, 184 
Distomum lanceolatum, 185 
Distomum pulmonale, 184 
Distomum pulmonale in sputum, 31 
Dittrich's plugs, 25 
Donne's test for pus, 353 
Dorrance's coagulometer, 98 
Dry heat sterilization, 408 
Dysentery bacillus in feces, 263 

agglutination of, 442 



E 



Earthy phosphates in urine, 284 
Echinococcus booklets lu sputum, 30 
Echinococcus cyst fluid, 398 
Ehrlich's benzaldehyd reaction, 377 
Ehrlich's triacid stain, 68 
Elastic tissue in sputum, 28 

staining of, 29 
EUermann-Erlandsen method, 37 
Enumeration of corpuscles, 56 



516 



INDEX. 



Enumeration of red cells, 59 

percentage of white cells, 61 
Eosin-hematoxylin stain, 70 
Eosinophils in sputum, 27 
Epithelial casts, 356 
Epithelia in urine, 353, 358 
Epizoa, 177 

Epstein's method for blood sugar, 129 
Esbach's tests for albumin, 323 

Tsuchiya's modification of, 325 
Estimation of blood corpuscles, 56 

counting diaphragm for, 64 

hematokrit, method of, 59 

Levy-Neubauer method of, 57 
Ethereal sulphates in urine, 287 
Ethylene diamin hydrate test for 

acetone, 343 
Ewald's method for peptic activity, 

228 
Ewald test-meal, 209 
Extrarenal albuminuria, 321 
Exudates, 391 

acetic acid test for, 394 

chemical characteristics of, 393 

cytology of, 397 

differential test for, 394 

Heller's test for, 394 

microscopic examination of, 396 

obtaiaing specimen of, 392 

pleural, 399 
physical characteristics of, 393 
varieties of, 395 
Eyepiece micrometer, 18 



Fatty acids in stomach, 224 

Fatty casts, 356 

Fat determination in milk, 404 

centrifugal method, 404 
Fats in feces, tests for, 252 
Feces, amylolytic activity of, 247 

bacterial examination of, 260 

blood in, 260 

chemical examination of, 249 

glutoid capsule test, 240 

inspection of, 243 

lost albumin in, 250 

macroscopic method, 246 

microscopic method, 244 

motor function^ of intestinal tract, 
241 

pancreatic insuflBciency, 246 

physical characteristics of, 239 

tubercle bacillus in, 427 

total fats in, 254 

urobilin in, 260 
Fehling's, qualitative method, 327 
Fehling's quantitative method, 332 
Feri's improved diazo-reaction, 365 
Fermentation test of feces, 251 

for sugar, 334 
Fibrin in urine, 326 
Filaria sanguinis hominis, 190 
Filling culture tubes, 422 
Fischer's test meal, 210 
Flagella, staining of, 434 
Flagellata, 181 

cercomonas intestinalis, 182 

trichomonas intestinalis, 182 

trichomonas vaginalis, 181 

trypanosoma, 182 
Flat-worms, 184 
Flea, 197 
Fleischl's method for hemoglobin, 52 

Meischer's method, 53 



Folin's method for total acidity, 281 

Folin and Denis's method for non- 
protein nitrogen, 112 

Folin-Farmer's method for total nitro- 
gen, 113, 313 

Folin-Schaffer's test for uric acid, 
306 

Formaldehyde in milk, 406 

Formalin test for ammonia in the 
urine, 315 

Fractional gastric analysis, 201 

Free gastric acidity, 205 

Free hydrochloric acid in stomach, 
218 
quantitative test for, 221 

Friedrich's test for free hydrochloric 
acid, 219 

Functional kidney test, 369 

Functional tests, cardiac, 151 
Graupner's, 152 
Schapiro's, 152 
work test, 152 



Gas bacillus in feces, 262 
Gastric acidity, quantitative estima- 
tion, 221 
Ga;stric analysis, 200 

absorption rate determination, 235 

acids in, 218 

bile in, 235 

bile-pigments in, 236 

capacity of stomach in, 215 

fasting stomach in, 217 

fractional method in, 201 

free acidity in, 205 

microscopic, 225 

motor functions in, 236 

occult blood in, 227 

organic acids in, 222 

outline of stomach in, 213 

pancreatic activity, 232 

peptic activity, 228 

preparation of patient for, 208 

removal of residuum of, 201 

rennin activity of, 234 

test meals for study of, 209 

total acidity of, 203 

tryptic activity of, 233 

x-ray examination of, 238 
Gastric lavage, 216 
Geraghty and Rowntree's test, 370 
Gerhardt's test for diacetic acid, 345 
Giemsa stain for treponema, 173 
Glucose in urine, 327 

Bang's test for, 333 

Benedict's test for, 332 

Bottger's test for, 330 

de Jager's test for, 329 

differential density test for, 336 

Fehling's test for, 327, 332 

fermentation test for, 334 

Haines's test for, 329 

Ny lander's test for, 336 

phenyl-hydrazine test for, 331 

Purdy's test for, 333 

Trommer's test for, 330 
Glutoid capsule test, 240 
Glycerin-agar, 419 
Glycosuria, 327 

Gmelin's test for bile pigments, 346 
Gmelin's test for bilirubin, 260 
Goldhorn stain, 173 
Gonococcal conjunctivitis, 371 
Gonococcus of Neisser, 429 



INDEX. 



517 



Gower's method for hemoglobin, 51 
Gram- negative organisms, 434 
Gram-positive organisms, 433 
Gram's staining method, 432 
Granular casts, 355 
Graupner's blood-pressure test, 152 
Gross's method for pancreatic activ- 
ity, 233 
Guaiac test for blood in stool, 255 
Gunning's test for acetone, 344 
Gunzberg's test for free hydrochloric 
acid, 218, 237 



Haines's test for glucose, 329 
Hammerschlag's method for peptic 

activity, 229 
Hawk's method for amylolytic activ- 
ity, 247 
Head louse, 195 

Heart-failure cells in sputum. 27 
Heart-failure cells in urine, 359 
Hecht-Weinberg-Gradwohl test, 458 

technic of, 459 
Hehner's test for formaldehyde, 406 
Heller's test for albumin, 322 
Heller's test for exudates, 394 
Hemocytometer, Levy-Neubauer, 57 
Hematin, 89 
Hematokrit, 60 
Hematoporphyrin, 91 
Hematuria, 350 
Hemoglobin, estimation of, 51 

Dare's method, 55 

Fleischl's method, 52 

Fleischl-Meischer's method, 53 

Gower's method, 51 
Hemoglobin, reduced, 89 
Hemoglobinuria, 352 
Hemolysis, 446 
Hemoptysis, 24 
Hemorrhagic exudates, 395 
Hemorrhagic sputum, 22 
Hemosporidia, 183 
Hess's method for capsules, 437 
Hippuric acid in urine, 308 

appearance of, 306 
Hirose test for liver function, 377 
Hodgkin's disease, 83 
Human milk, 400, 402 

collecting sample of, 402 

composition of, 405 

examination of, 403 

fat determination! of, 404 

microscopic appearance of, 403 

physical characteristics of, 402 

quantity of, 403 

reaction of, 403 

specific gravity of, 403 

sugar determination in, 405 
Hyalo-granular casts, 356 
Hydrocele fluid, 401 
Hydrochloric acid, 217 

qualitative estimation of, 218 

quantitative estimation of, 221 
Hydrochloric acid test, 237 

Gunzberg's method, 237 

Sahli's method, 237 
Hydrogen-ion concentration, 118 

significance of, 121 
Hydruria, 275 
Hypertension, 151 

in life insurance examination, 153 

transient albuminuria in, 153 
Hypotension, 151 

in life insurance examination, 153 



Illumination for microscope, 6 
Incubator, the, 424 

thermo regulator for, 425 
India-ink method, 174 
Indican, 287 

Jaffe's test for, 287 

Obermayer's test for, 288 

quantitative test for, 288 
Indol-acetic acid, 291 
Inflation of stomach, 213 

contraindications to, 214 
Inorganic constituents of urine, 282 
Inorganic urinary sediments, 361 

acid group, 362 

alkaline group, 363 
Intermittent sterilization, 412 
lodin in urine, 367 

Iodoform test for motor function, 237 
lodophilia, 85 

Iris diaphragm for microscope, 4 
Itch parasite, 194 
Ives diffraction method, 92 



Jaffe's test for creatinin, 309 
Jaffe's test for indican, 287 
Jaquet's polygraph, 155 



K 



Kala-azar, 171 

Kelling's test for lactic acid, 224 
Kjeldahl's method for total nitro- 
gen, 113, 311 
KlUnge's test for blood in stool, 256 
Koch-Weeks bacillus, 391 
Kolb's stain, 174 



Lactic acid in stomach, 223 

Lactosuria, 342 

Lange's colloidal gold test, 386 

Lead in urine, 367 

Legal's test for acetone, 343 

Leishmann-Donovan bodies, 171 

occurrence of, 171 

staining of, 171 
Leo's method for estimating rennin 

activity, 234 
Lepra bacillus, 41 
Leptus autumnalis, 195 
Leucin in urine, 318 
Lieben's test for acetone, 344 
Liebermann's test for formaldehyde in 

milk, 406 
Life insurance examinations, blood- 
pressure in, 153 
Liver fluke, 184 
Liver function test, 377 
Loffler's blood serum mixture, 422 
Loffler's flagella staining, 434 
Lohnstein's saccharimeter, 336 
Tx>st albumin test of feces, 250 
Luetic antigen, 447 
Luetin reaction of Noguchi, 460 

specificity of, 462 

technic of, 461 
Leukocytic leukemia, 82 
Leukemia, 81 

leukocytic, 82 

lymphatic, 82 
Leukocyte count, 15, 63 



518 



INDEX. 



Leukocyte count In meningitis, 382 
of spinal fluid, 3^ 
in syphilis, 382 
in tuberculosis, 382 
Leukocytes, varieties of, 71 
chart for counting, 73 
differential count of, 72 
Leukocytosis, 77 
physiologic, 77 
pathologic, 77 
Levulose in urine, 341 

test for, 341 
Levy, Rovrntree and Marriott's 

method, 121 
Lewis and Benedict's method for 

blood sugar, 131 
Lymphatic leukemia, 82 



M 



McCaskey's method for viscosity, 104 
Mackenzie's polygraph, 157 
Macroscopic Widal reaction, 441 
Malarial parasites, 159 

appearance of, 69 

cultivation of, 163 

d-etection of, 161 

staining methods, 161 
varieties of, 161 
Malta fever agglutination test, 443 
Maltosuria, 342 
Maltwood finder, 11 

method of using, 10 
Marriott's method for carbon-dioxide 

tension, 125 
Marshall's method for blood urea, 107 

urinary urea, 299 
Mean blood-pressure, 135 
Mechanical stage, 10 
Medicinal discoloration of urine, 272 
Meiostagmin reaction, 463 
Meirowsky's stain, 175 
Mendelbaum's test for typhoid fever, 

443 
Meningitis, acute, cell count in, 382 

syphilitic, cell count, 382 

tubercular, cell count, 382 
Meningococcus, 430 
Mercury in urine, 367 
Methemoglobin, 89 

Methyl-violet test for free hydro- 
chloric acid, 219 
Methylene-blue reaction in feces, 366 

for bile pigments, 347 
Mett's method for peptic activity, 230 

for pancreatic activity, 233 

for urinary solids, 279 
Micrococcus lanceolatus, 39 
Micrococcus meningitidis, 430 
Microscope, apochromatic objective, 5 

adjustments of, 4 

barrel of, 3 

blood examinatioru, 15 

camera lucida for, 16 

care of, 1, 9 

dark-ground illumination, 7 
improvised dark-ground illumina- 
tion, 8 

description of, 2 

draw tube of, 4 

eye-piece micrometer for, 18 

eye piece of, 2 

illumination, 6 

iris diaphragm, 4 

maltwood finder for, 11 

mechanical stage for, 10 



Microscope, micrometer for, 17 

micrometer stage for, 17 

nose piece of, 4 

objectives of, 3 

oblique light, 7 

ocular of, 2 

oil-immersion objective, 5 

reflector for, 4 

stage of, 3 

substage condenser, 4 

urinary sediments, 13 

■warm stage for, 11 
Microscopic examination of blood, 65 

of feces, 244 

of gastric contents, 225 

of pleural fluid, 400 

of saliva, 389 

of Widal reaction, 439 
Microsporon furfur, 199 
Microsporon Audouini, 199 
Milan's method for coagulation, 97 
Milk, 400 

formaldehyde in, 406 
Motor function of stomach, 236 

iodoform test for, 237 

Boas's test for, 237 
Motor function of intestines, 241 

Schmidt's method for, 241 
Much's staining method, 38 
Muir's method for staining capsules, 

436 
Murexid test for uric acid, 303 
Myer's method for cholesterol, 114 
Myocarditis, blood-pressure in, 151 

functional tests for, 151 



N 



Nakayama's test for bile pigments. 

Nasal secretions, 391 
Xeisser's coccus, 429 

cultivation of, 429 
Nematodes, 189 
Neuman's ocein test, 342 
Nitric acid test for bile pigments, 347 
Nitrogen, total in urine, 311 
Nitrogenous equilibrium, 295 
Noguchi's butyric acid test for pro- 
tein, 3S4 
Noguchi's leutin reaction, 460 

culture method, 176 

staining method, 176 

Wassermann reaction, 449 
technlc of reaction, 450 
value of reaction, 457 
Nonne's test, 385 
Non-protein nitrogen in blood, 112 

Folin and Dennis's method, 112 

Taylor and Hulton's method, 112 
Normal differential count, 74 
Nucleo-albumin, 326 
Nutrient gelatin, 418 
Nylander's bismuth test, 330 



Obermayer's test for indican, 288 

Objective of microscope, 3 

Oblique light, 6 

Occult blood in stomach contents, 227 

benzidin test, 228 

Boas's test for, 227 

guaiac test for, 227 

orthotolidin test. 228 
Occult blood in stool, 254 



INDEX. 



519 



Occult blood In urine, 351 

Ocular of microscope, 2 

Oidium albicans, 390 

Oil immersion objective, 5 

Oliguria, 275 

Oliver's test for bile acids, 349 

Oppenheim and Sach's stain, 173 

Opsonic method, 464 

Oral secretions, 387 

diphtheria bacilli in, 389 

microscopic examination, 389 

oidium albicans in, 390 

pancreatic function, 388 

ptyalin content of, 388 

Vincent's spirillum in, 390 
Organic gastric acids, 222 
Orthotolidin test for occult blood, 228 
Osmond's blood count form, 73 
Outlining the stomach, 213 
Ovarian cyst fluids, 398 
Oxalic acid in urine, 310 

appearance of, 310 

quantitative determination of, 310 
Oxalates in urine, 363 
Oxybutyric acid in urine, 346 
Oxyhemoglobin, 89 
Oxyuris vermicularis, 193 



Pacini's color reaction, 42 

Palpatory method of blood-pressure, 

142 
Pancreatic activity of gastric juice, 
232 

Gross* method, 233 

Mett's method, 233 
Pancreatic cyst fluid, 399 
Pancreatic function test, 388 
Pancreatic insufficiency, Schmidt's test 

for, 246 
Pappenheim's stain, 33 
Parasites, 159 

filaria, 165 

Leishmann-Donovan bodies, 171 

malarial, 159 

scarlet fever, 163 

spirilla of Obermeier, 169 

treponema pallidum, 172 

trjrpanosomes, 167 

yellow fever, 165 
Parasites, animal, 177 
Parasites in stools, 244 
Paramecium coli, 184 
Paratyphoid agglutination, 441 
Pathologic stools, 243 
Pearce's method for blood sugar, 133 
Pediculus capitis, 195 

pubis, 196 

vestementi, 195 
Pentosuria, 342 

Newmann's test, 342 
Penzoldt's method, 235 
Peptic activity, 228 

Ewald's method for, 228 

Hammerschlag's method for, 229 

Mett's method for, 230 
Peptone in gastric contents, 225 
Percentage of erethrocytes, 61 
Pericardial fluid, 401 
Peritoneal fluid, 397 

hemorrhagic, 398 

inflammatory, 398 

milky, 398 

serous, 398 
Pernicious anemia, 79 



Pertussis bacillus, 42 

Petri dishes, 424 

Pettenkofer's test for bile acids, 349 

PfeifCer's bacillus, 40 

Phenolphthalein test for blood in 

stools, 258 
Phenolphthalein test for kidney func- 
tion, 369 
Phenylhydrazin test, 331 
Phosphates in urine, 283, 363 
Phthiriasis, 196 
Pineapple test, 224 
Pink eye, 391 
Plague agglutination, 442 
Plasma, blood, alkaline reserve of, 
118 

Sorensen's color standards, 122 
Plasmodium of malaria, 159 
Plate making, 423 
Platyhelminthes, 184 
Pleural fluid, inflammatory exudates, 
400 

microscopic appearance of, 400 

non-inflammatory, 399 

purulent, 400 
Polariscopic estimation of glucose in 

urine, 337 
Polygraph, the, 155 
Polymorphonuclear leucocytes, classi- 
fication of, 75 
Polyuria, 275 

Preservation of urine sample, 270 
Pressure, arterial, 134 

cerebrospinal, 380 

pathologic modifications, 381 

physiologic modifications, 381 
Primary anemias, 79 
Progressive pernicious anemia, 79 
Protein content, spinal fluid, 384 

Noguchi's butyric acid test for, 384 

Ross Jones's test for, 384 
Protein determination in milk, 405 
Proteose in urine, 326 
Protozoa, 179 
Protozoa in feces, 260 
Pseudoleukemia, 83 
Pseudoreactions, 465 
Ptyalin In saliva, 388 
Pulex irrltans, 197 
Pulex penetrans, 197 
Pulse, the, 134 
Pulse pressure, 134, 145 

significance of, 136 
Pulse tracings, 157 
Purdy's test for albumin, 323 

for chlorides, 292 

for glucose, 333 
Purins in the urine, 307 
Purulent exudates, 395 

pleurisy, 400 
Pus cells in sputum, 26 

in stools, 243 

in urine, 352, 359 
Putrid exudates, 396 
Pycnometer, the, 47 
Pyuria, 352 



Qualitative test for albumin, 321 

acetic acid, 321 

Heller's, 322 

Purdy's, 323 

Tanret's, 323 

Ulrich's, 323 
Qualitative test for glucose, 327 



520 



INDEX. 



Qualitative test for glucose, Bottger's 
bismuth, 330 

de Jager's, 329 

Fehling's, 327 

Haines's, 329 

Nylander's, 330 

phenylhydrazin, 331 

Trommer's, 330 
Qualitative test for uric acid, 303 
Quantitative estimation of ammonia 

in urine, 314 
Quantitative estimation of casts, 358 
Quantitative estimation of oxalic acid, 

310 
Quantitative tests for albumin, Es- 
bach's, 323 

Tsucbiya's, 325 
Quantitative tests for glucose, 332 

Bang's, 333 

Benedict's. 332 

differential density, 336 

Fehling's, 332 

fermentation, 334 

polarimetric, 337 

Purdy's, 333 
Quantitative test for indican, 288 
Quantitative tests for uric acid, 301 

Folin-Shaffer's method for, 306 

Ruhrmatm's method, 304 



Range of blood-pressure, 134 
Reaction of the blood, 48 
Reaction of feces, 239, 249 

fermentation test for, 251 

sublimate test for, 250 
Reaction of urine, 279 
Reaction, Diazo, 365 

Noguchi's, 449 

Wassermann, 445 
Reagent, Sahli's, 205 
Recording blood-pressure, 146 
Red blood cell count, 59 
Reduced hemoglobin, 89 
Reduction tests for glucose, 327 
Reflector of microscope, 4 
Rehfus's fractional gastric method, 201 
Relapsing fever, 169 

organism of, 169 

staining of, 170 
Renal capacity test, 369 
Rennin activity of stomach, 234 

Leo's method for, 234 
Retention meal, 202 
Rhizopodia, 180 

ameba histolytica, 180 
Riegel's test meal, 210 
Ritter's albumin test, 43 
Rivalta's acetic acid test, 44 
Romano wski staining method, 68 
Rose and Coleman's method for urea 

in blood. 111 
Rosenbach's test for bile pigments, 

348 
Rosenbach's test for skatol, 291 
Rosenberger's technic for tubercle 

bacillus, 263 
Ross Jones's test, protein, 384 
Ruhemann's test for uric acid, 304 
Russel and Brodie's method for coag- 
ulation time, 97 
Russo's test for urine, 366 



S 



Saccharimeter, fermentation, 334 

Lohnstein's, 336 
Sahli's desmoid reaction, 237 
Sahli's reagent, 205 
Saliva, 389 

Salkowskl-Volhard test, 292 
Salzer's test meal, 211 
Santonin in urine, 369 
Sarcoptes scabiei, 177, 194 
Saxon's method for total fats, 252 
Scarlet fever, 163 

organism causing, 163 
Schapiro's blood-pressure test, 152 
Schick test for diphtheria, 464 

specificity of, 466 

technic of, 467 
Schiff's test for uric acid, 303 
Schlesinger's test for urobilin, 260 
Schlosing's test for ammonia, 314 
Schmidt's method for feces, 241 

bilirubin, 260 

pancreatic function, 246 

method for urobilin, 260 
Secretion of hydrochloric acid, 217 
Secretions, conjunctival, 391 

nasal, 391 

oral, 387 
Sediments, urinary, 13 
Seliwanoff's test for levulose, 341 
Sero-diagnosis, 438 

agglutination reactions, 438 
Sero-fibrinous effusion, 400 
Serous stools, 239 
Serum albumin in urine, 326 
Serum globulin in urine, 326 
Sheep cells, preparation of, 454 
Shiga's bacillus, 442 
Significance of glycosuria, 341 
Skatol in urine, 291 

amylic alcohol test, 291 

Rosenbach's test for, 291 
Smith's test for bile pigments, 347 
Sorensen's color standards, 122 
Specific blood reactions, 438 
Specific gravity of blood, 47 

of milk, 403 

of urine, 275 
Specimen of blood for examination, 50 
Spectroscopic examinations, 87 

alkaline hematin, 91 

chlorophyl, 90 

CO-hemoglobin, 90 

cyan-hemoglobin, 90 

hematin, 89 

hematoporphyrin, 91 

hemoglobin, 89 

Ives diffraction method for, 92 

methemoglobin, 89 

oxyhemoglobin, 89 

principle of, 88 

spectroscope, the, in, 87 

urobilin, 90 

uroroseinogen, 91 
Spectroscopic test for blood in stool, 

259 
Spencer's method for tryptic activity, 

233 
Spengler's method, 38 
Spermaturia, 360 
Sphygmograph, the, 154 

Jacquet's polygraph, 155 

Mackenzie's polygraph, 157 
Sphygmography, 154 

normal pulse curve, 157 



INDEX. 



521 



Sphygmomanometer, 136 

operation of, 141 

types of, 137 
aneroid, 140 
mercury, 137 
Sphygmomanometry, 134 
Spirochaeta pallida, 172 
Sporozoa, 183 

coccidium hominis, 18'i 

hemosporidise, 183 
Sputum, the, 19 

actinomyces in, 30 

air current of, 23 

albumin content of, 43 

antiformin treatment of, 35 

bacteria in, 40 
of influenza, 40 
of leprosy, 41 

of micrococcus lanceolatus, 39 
of pertussis, 42 
of tuberculosis, 31 

bacterial content of, 34 

capsule staining of bacteria in, 40 

casts in, 21 

chemical characteristics of, 19 

color of, 21 

concretions in. 25 

crystals in, 28 

Curschman's spirals in, 27 

distomum pulmonale in, 31 

Dittrich's plugs in, 25 

echinococcus booklets in, 30 

elastic fibers in, 28 

Ellermann-Erlandsen method, 37 

eosinophiles in, 27 

heart-failure cells in, 27 

hemorrhagic, 22 

macroscopic appearance of, 20 

microscopic examination of, 26 

Much's method for, 38 

odor of, 24 

physical characteristics of, 19 

pus cells in, 26 

Racini's color reactioa of, 42 

Hitter's albumin test for, 43 

Rivalta's acetic acid test, 44 

special reactions of, 42 

S'pengler's method for, 38 

stained specimen of, 31 
Stage of microscope, 3 
Stain for capsules, 436 
Staining bacteria in body fluids, 436 

Hiss's method for capsules, 437 
Staining of flagella, 434 
Staining methods for elastic tissue in 
sputum, 29 

Czaplewsky's method, 33 

for filaria, 167 

for spirillse, 170 

Giemsa's, 173 

Goldhorn's, 173 

india-ink, 174 

Kolb's, 174 

for Leishmann-Donovan bodies, 171 

for malarial parasites, 161 

for trypanosomes, 169 

Mierowsky's, 175 

Oppenheim and Sachs's, 173 

Pappenheim's method, 33 

treponema pallidum, 173 

"Woods's, 173 

for tubercle bacillus, 31 

Ziehl-Nielsen method, 32 
Starch digestion in stomach, 225 
Steam sterilization, 410 



Sterilization by, 407 

Arnold sterilizer, 414 

autoclave, 412 

chemicals, 415 

dry heat, 408 

intermittent method, 412 

steam, 410 
Stomach functions, 200 
Strauss's test for lactic acid, 223 
Streptococcus in feces, 262 
Strongyloides intestinalis, 189 
Sublimate test, 250 
Substage condenser, 4" 
Sugar-agar, 420 

Sugar determination in milk, 415 
Sulphates in urine, 286, 363 
Synovial fluid, 401 
Syphilis, 172 
Systolic blood-pressure, 134, 142 

auscultatory method, 143 

palpating method, 142 



Tannin in urine, 369 
Tanret's test for albumin, 323 
Taylor and Hulton's method for non- 
protein nitrogen, 112 
Technic of Wassermann reaction, 446 
Temporary parasites, 194 
arthropoda, 194 
arachnoidae, 194 
Tenia echinococcus, 188 
Tenia lanceolata, 187 
Tenia lata, 186 
Tenia nana, 186 
Tenia saglnata, 188 
Tenia solium, 187 

Tests for acetic acid in gastric con- 
tents, 224 
acetoacetic acid in urine, 344 
acetone in urine, 343 
ethylene-diamin hydrate, 343 
Gunning's, 344 
Legal's, 343 
Lieben's, 344 
Trommer's, 343 
albumin in urine, 322 
Esbach's, 323 
Heller's, 322 
Purdy's, 323 
Tanret's, 323 
Tsuchya's, 325 
Ulrich's, 322 
alcohol, 225 
bile, 235 

bile pigments, 236 
bilirubin, 349 
butyric acid, 224 
fatty acids, 224 

fluid absorption from stomach, 235 
free hydrochloric acid, 218 
glucose, Bang's quantitative. 333 
Benedict's quantitative, 332 
Bottger's qualitative, 330 
de Jager's qualitative, 329 
Fehling's, .qualitative, 327 
Fehling's quantitative, 332 
fermentation, 334 
Haines's qualitative, 329 
Nylander's qualitative, 330 
phenylhydrazin, 331 
Purdy's quantitative, 333 
Trommer's qualitative, 330 
hematuria, 350 



522 



INDEX. 



Tests for indican, 287 

lactic acid, 223 

motor function of stomach, 236 

occult blood in stomach contents, 
227 
in feces, 254 

pancreatic activity, 232 

pentose in urine, 342 

peptic activity, 228 

pus in urine, 353 

renal activity, 369 
value of, in surgery, 375 

rennin in gastric contents, 234 

starch in gastric contents, 225 

total gastric acidity, 220 

tryptic activity of gastric contents, 
233 

urea, 298 
quantitative, 299 

urobilin, 349 
Test meals, 209 

Boas's, 210 

Ewald's, 209 

Fischer's, 210 

Riegel's, 210 

Salzer's, 211 
Test-meal extraction, ^2 

Daland-Faught method, 206 

Rehfus method, 201 

Turck's double tube method, 212 
Test diet, Schmidt's, 241 
Thermo-regulator, 425 
Thrush, 390 
Titre of antigen, 455 
Tone-phases, 143 
Topfer's test for free hydrochloric, 

218 
Total acidity of urine, 281 

factors affecting, 281 

Folin's method, 281 
Total gastric acidity, 203, 220 

quantitative tests, 220 
Total nitrogen of blood, 113 

Folin-Farmer's method, U3 

Kjeldahl's method, 113 
Total nitrogen in urine, 311 

Folin-Farmer's method for, 313 

Kjeldahl's method for, 311 
Total phosphoric acid in uriae, 285 
Total solids in urine, 279 
Trichina spiralis, 191 
Trachoma, 391 
Transient albuminuria, 153 
Transudates, 391 

characteristics of, 393 

chemical characteristics of, 393 

cytology of, 397 

differential test, 394 

microscopic examination of, 396 

obtaining specimen of, 392 
Trapp's formula for total solids, 279 
Trematodes, 184 
Treponema pallidum, 172 

collection of specimen, 172 

culture of, 176 
Noguchi's method, 176 

dark field for, 7 

staining of, 173 

tissue staining for, 176 

vital staining of, 175 
Trichocephalalus dispar, 191 
Trichomonas vaginalis, 181 
Trichophyton, 198 
Trommer's test for acetone, 343 
Trommer's test for glucose, 330 
Tropeolin test for free HCl, 219 



Trypanosomes, 167 
T. gambiense, 168 
description of, 168 
isolation of, 168 
staining of, 169 
Tryptic activity, 233 

Spencer's method for, 233 
Tsuchiya's test for albumin, 325 
Tlibe-casts, 353 

varieties of, 355 
Tubercle bacillus, antiformin method 
for, 35 
granular forms of, 34 
Much's stain for, 38 
Rosenberger's method for, 427 
in blood, 427 
in feces, 427 
Spengler's method, 38 
sputum concentration for, 34 
Tubercle bacilli in cerebrospinal 
fluid, 387 
feces, 263 
sputum, 31 
Turck's double gastric tube, 212 
Twenty-four hours' urine specimen, 

268 
Typhoid agglutination test, 438 
Typhoid bacillus, 430 

blood cultures of, 431 
Typhoid fever, Mendelbaum's test for, 

443 
Tyrosia in urine, 318 

U 

Uffelman's test for lactic acid, 223 
Ulrich's test for albumin, 323 
Uncinaria americana, 192 
Universal staining method, 68 
Urates in urine, 307, 362 

qualitative test for, 307 

microscopic appearance of, 308 
Urea in body fluids, 111 

in blood, 107 
Urea in urine, 294 

detection of, 298 

estimation of, 298 

in disease, 2S7 

normal equilibrium of, 295 

properties of, 297 
Uric acid in urine, 302, 362 

isolation of, 303 

murexid test for, 303 

properties of, 302 

Schiff's test for, 303 
Urinalysis, 268 
Urinary concretions, 364 

discolorations, 271 

sediments, 13, 354 
Urine, examinations for, acetic acid, 
344 

aceto-acetic acid, 344 

acetone, 343 

albumin, 319 

ammonia, 314 

amount of, 273 

bile acids in, 348 

bile-pigments in, 346 

bile in, 346 

Cammidge reaction*, 376 

casts, 353 

chlorides, 292 

creatin, 309 

creatinin, 309 

concretions, 364 

cylindroids, 357 



INDEX. 



523 



Urine, examination for, cystin, 317 

decomposition changes, 269 

diacetic acid, 345 

diazo reaction, 365 

epithelial cells, 353 

erythrocytes, 352 

functional kidney activity, 369 

general characteristics, 270 

glucose, 327 

hemoglobinuria, 352 

hippuric acid, 308 

indican, 287 

inorganic sediment, 361 

leucin, 318 

leucocytes, 77 

levulose, 341 

metallic poisons, 367 

non-protein nitrogen, 311 

organic sediment, 293, 359 

oxalic acid, 310 

oxybutyric acid, 346 

phosphates, 383 

physical characteristics, 271 

proteoses, 326 

purins, 307 

pus cells, 352 

reaction, 279 

specific gravity, 275 

sulphates, 286 

total acidity, 281 

total nitrogen, 311 

total solids, 279 

tyrosin, 318 

urates, 307 

urea, 294 

uric acid, 302 

urobiUn, 349 
Urinometer, 276 
Urobilin in feces, 90, 260 

tests for, 349 
Uroresin in urine, 291 
Uroroseinogen, 91 



Van Deen's test for blood in stool, 255 
Van Slyke and Cullins's method for 
urea in blood, HO 



Van Slyke and Cullins's method for 

urea in urine, 301 
Vegetable parasites, 197 

achorion Schonleinii, 197 

microsporon Audouini, 199 

microsporon furfur, 19d 

trichophyton, 198 
Vincent's angina, 390 
Viscosity of the blood, 102 

factors affecting, 103 

methods of determining, 104 

McCaskey's method, 104 

Watson-Dunning's method, 106 
Vital staining of blood, 84 
Volume index of erythrocytes, 62 
Von der Velden's test for free HCl, 
219 



W 



Warm stage for microscope, 11 
Waxy casts, 357 
Wassermann reaction, 445 

luetic antigen, 447 

technic of, 447 
Weber's spectroscopic test, 259 
Welsh's method for staining capsules, 

436 
Westphal balance, 278 
Weyl's test for creatinin, 309 
Widal reaction, 438 

macroscopic reaction, 441 

microscopic reaction, 440 
Wolgemuth's method for amylolyUc 

activity, 247 
Wood's stain, 173 
Wright's differential stain, 433 



X-ray examination of stomach, 238 

Y 
Yellow fever, 165 



Ziehl-Nielsen method, 32 



